Biomass conversion to fuels and chemicals

ABSTRACT

This disclosure relates to compositions and methods for converting biomass to various chemical intermediates and final products including fuels. Aspects include the depolymerization of lignin, cellulose, and hemicellulose to a wide slate of depolymerization compounds that can be subsequently metabolized by genetically modified bacterium, and converted to cis,cis-muconic acid. Other aspects include the use of monometallic catalysts for converting the cis,cis-muconic acid to commodity chemicals and fuels, for example adipic acid and/or nylon.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. Provisional Application No. 62/026,222, filed on Jul. 18, 2014, and U.S. Provisional Application No. 62/062,224, filed on Oct. 10, 2014, both entitled “Biomass Conversion to Fuels and Chemicals,” and both of which are incorporated herein by reference in their entireties.

CONTRACTUAL ORIGIN

The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronic text file entitled “14-04_ST25.txt,” having a size in bytes of 141 kb and created on Jul. 20, 2015. Pursuant to 37 CFR §1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.

BACKGROUND

Lignocellulosic biomass represents a vast resource for the production of renewable transportation fuels and chemicals to offset and replace current fossil fuel usage. For many decades, worldwide research efforts have focused on the development of cost-effective processes to selectively convert the polysaccharide components of plant cell walls, namely cellulose and hemicellulose, to fuels and chemicals through biological and chemical pathways. For example, in bioethanol production, biomass typically undergoes a mild thermochemical pretreatment step followed by enzymatic hydrolysis and fermentation to produce ethanol from the monomeric components of both cellulose and hemicellulose.

The lignin component of lignocellulosic biomass is an energy-dense, heterogeneous alkyl-aromatic polymer constructed from phenylpropanoid monomers used by plants for water transport and defense, and it is the second most abundant biopolymer on Earth after cellulose. Lignin is typically underutilized in most selective conversion processes for biofuel production. In the production of fuels and chemicals from biomass, lignin is typically burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively upgrade the monomers to value added products. This limited ability to utilize lignin, despite being the most energy dense polymer in the plant cell wall, is primarily due to its inherent heterogeneity and recalcitrance. Despite having a longer history of use as alternative renewable raw materials, cellulose and hemicellulose still remain important, high volume, readily available renewable raw materials, and next generation technologies that process these polysaccharides efficiently and economically are still needed. Thus, compositions, methods, and processes that can simultaneously and/or in parallel convert all of the substituent components of biomass, e.g. lignin, cellulose, and hemicellulose, to useful chemical intermediates, final chemical products (including fuels), is highly desirable to make steps towards lessening global dependency on petroleum.

However, in order to displace our current petrochemical consumption, an expanded renewable product slate is necessary, similar to the myriad of products currently derived from crude petroleum. This requires efficient and economically viable technology for converting all of the main constituents of biomass, cellulose, hemicellulose, as well as lignin, to useful final products, as well as chemical intermediates that can be converted to useful final products, utilizing either new technologies or existing technologies. The present application provides a suite of innovative technologies that may serve as cornerstones for future biomass-to-chemicals manufacturing plants, wherein these technologies focus on the first task of converting biomass to cis, cis-Muconic acid (hereinafter referred to as “muconic acid”), followed by the second task of converting the muconic acid to useful products including, but not limited to, adipic acid, 1,6-hexanediol, and hydrocarbon fuels.

Genetic engineering of microbial organisms is most commonly known due to the landmark Supreme Court case of Diamond v. Chakrabarty, wherein the court validated Chakrabarty's U.S. Pat. No. 4,259,444, directed to a Pseudomonas putida strain that had been engineered to degrade various oil derivatives, including octane and naphthalene.

Since then, researchers have pursued engineered microorganisms for biologically converting various biomass components to numerous chemical intermediates and products, including muconic acid, followed by conversion to adipic acid. Annual world-wide production of adipic acid in 1989 was estimated at 4.2 billion pounds and production has continued to grow since then. With U.S. production at 1.75 billion pounds in 1992, adipic acid consistently ranks as one the top fifty chemicals produced domestically. Nearly 90% of domestic adipic acid is used to produce nylon-6,6. Other uses for adipic acid include production of lubricants and plasticizers. Thus, there is a large economic driver behind the development of improved methods for muconic acid production, especially for the development of improved production methods that utilize renewable resources.

For example Koppisch et al. (“Koppisch”) describe the use of engineered prokaryotic organisms for converting D-glucose to catechol and muconic acid (WO 2012/106257). This includes the introduction of exogenous decarboxylase genes, including aroY from Klebsiella pneumoniae, and the introduction of exogenous dioxygenase genes for converting catechol to muconic acid, for example catA.

U.S. Pat. No. 5,487,987 to Frost et al. (“Frost”) describes the production of adipic acid through a metabolic pathway producing the cis, cis-muconic acid intermediate, also utilizing D-glucose as the starting material, and Escherichia coli genetically engineered to include genes endogenous to Klebsiella pneumoniae and Acinetobacter calcoaceticus.

Burk et al. (“Burk”) describes the use of engineered microbial microorganisms to produce terephthalate through a muconic acid intermediate comprising trans,trans-muconate and/or cis, trans-muconate, starting with succinyl-CoA as a starting material (WO 2011/017560).

U.S. Pat. No. 8,133,704 to Baynes et al. (“Baynes”) describes the use of genetically engineered microorganisms including E. coli, C. glutanicum, B. flavum, and B. lactofermentum for the eventual production of adipic acid, utilizing carbohydrate starting materials.

Weber et al. describe a genetically modified Saccharomyces cerevisiae to produce cis, cis-muconic acid utilizing aromatic amino acid pathways (Applied and Environmental Microbiology (2012) 78(23), 8421-8430).

Pseudomonas putida has been of particular interest recently, especially since completion of the genomic sequencing of Pseudomonas putida KT2440 (Environmetal Microbiology (2002) 4(12), 799-808). Jimenez et al. have characterized four of the main pathways in the KT2440 strain, including the protocatechuate and catechol branches of the β-ketoadipate pathway, the homogentisate pathway, and the phenylacetate pathway (Environmental Microbiology (2002) 4(12), 824-841).

Even before its genomic sequencing, scientists attempted to use P. putida as an organism for producing muconic acid. For example, U.S. Pat. Nos. 4,480,034 and 4,731,328 describe converting toluene to muconic acid, utilizing engineered microorganisms including Pseudomonas putida.

More recently, Bang et al. (“Bang”) describe the use of a P. putida strain (BM014) for the production of cis, cis-muconic acid utilizing benzoic acid as a starting material (Journal of Fermentation and Bioengineering (1995) 79(4), 381-383). J. van Duuren et al. describe the use of P. putida KT2440 for the production of cis, cis-muconic acid utilizing benzoate as a starting material (Journal of Biotechnology (2011) 156, 163-172).

Thus, a review of the literature illustrates that a significant need remains for improved, flexible, reliable, economical technologies that are capable of converting a wide variety of biomass to industrially relevant chemical intermediates and final products, especially technologies that are capable of converting all of the key constituents of biomass; e.g. lignin, cellulose, and hemicellulose. To achieve this goal, robust genetically modified microorganisms, and/or mixtures of microorganisms are required that are capable of funneling chemical compounds through multiple metabolic pathways to common a common precursor or precursors, that can be subsequently converted to useful chemical intermediates and final products. In addition, novel upstream and downstream processing techniques are needed to assist with biomass fractionation, lignin and polysaccharide depolymerization, and precursor conversion to chemical intermediates and final products. The concepts presented herein provide some technologies that address these and other needs.

SUMMARY

An aspect of the present invention is a genetically modified microorganism that includes at least one exogenous gene addition, wherein the at least one added gene encodes at least one of a decarboxylase, a dehydratase, or a monooxygenase. In some embodiments of the present invention, a genetically modified microorganism may have at least one deleted gene that encodes at least one of a dioxygenase, a muconate lactonizing enzyme, or muconolactone isomerase. In some embodiments of the present invention, a microorganism may over-express at least one demethylase gene. In some embodiments of the present invention, a microorganism may include a deletion of at least one catabolite repression control gene.

In some embodiments of the present invention, the at least one exogenous gene may encode a decarboxylase from Enterobacter cloacae. In some embodiments of the present invention, the exogenous gene may be at least one of aroY, ecdB, or ecdD. In some embodiments of the present invention, the at least one exogenous gene may encode a dehydratase from Bacillus cereus or from P. pneumonia. In some further embodiments of the present invention, the exogenous gene may be at least one of aroZ or asbF. In some further embodiments of the present invention, the at least one exogenous gene may encode a monooxygenase from Pseudomonas putida CF600. In still further embodiments of the present invention, the exogenous gene may be at least one of dmpK, dmpL, dmpM, dmpN, dmpO, dmpP, or pheA. In still further embodiments of the present invention, the at least one deleted gene may be at least one of pcaH or pcaG. In some embodiments of the present invention, the at least one deleted gene from a microorganism may be at least one of catB or catC. In some embodiments of the present invention, the demethylase gene may be at least one of vanA, vanB, or ligM.

In some embodiments of the present invention, the microorganism may be at least one of a fungi, a prokaryote, or a prokaryotic microorganism. In some embodiments of the present invention, the microorganism may be a prokaryote or prokaryotic microorganism from the genus Pseudomonas. In some embodiments of the present invention, the microorganism may be a strain of P. putida, P. fluorescens, or P. stutzeri. In some further embodiments of the present invention, the microorganism may be a strain of P. putida KT2440.

A further aspect of the present invention is a process for producing muconic acid, where the process includes contacting a culture broth containing lignin depolymerization compounds with any of the genetically modified microorganism disclosed within this specification. In some embodiments of the present invention, the lignin depolymerization compounds may include at least one of p-coumaric acid, ferulic acid, benzoic acid, phenol, coniferyl alcohol, caffeic acid, vanillin, or 4-hydroxybenzoic acid, and at least a portion of the lignin depolymerization compounds are converted to catechol, and at least a portion of the catechol is converted to muconic acid.

A further aspect of the present invention is a process for producing adipic acid, where the process includes separating muconic acid from a culture broth comprising muconic acid, impurities, and microorganisms, purifying the separated muconic acid, and hydrogenating at least a portion of the purified muconic acid to produce the adipic acid or other chemicals. In some embodiments of the present invention, the separating may include at least one of centrifugation and/or filtration to produce muconic acid that is substantially free of the microorganism. In some embodiments of the present invention, the purifying may include contacting the separated muconic acid with an adsorbent, wherein the adsorbent removes at least a first portion of the impurities from the separated muconic acid.

In some embodiments of the present invention, the adsorbent may include activated carbon. In some embodiments of the present invention, the impurities removed may include at least one of benzoic acid, protocatechuic acid or 4-hydroxybenzoic acid. In some embodiments of the present invention, the purifying may include crystallizing at least a portion of the muconic acid from the separated muconic acid to form a muconic acid precipitate and a liquid that contains at least a portion of the impurities.

In some embodiments of the present invention, the purifying may include dissolving the muconic acid precipitate in a solvent, resulting in a liquid phase that includes muconic acid and a solid phase that includes at least a portion of the impurities, and separating the liquid phase from the solid phase. In some embodiments of the present invention, the separating may be by at least one of filtration or centrifugation. In some embodiments of the present invention, the hydrogenation may include contacting the liquid phase that includes muconic acid and diatomic hydrogen with a metallic catalyst. In some embodiments of the present invention, the metallic catalyst may include at least one of palladium, platinum, ruthenium, or rhodium. In some embodiments of the present invention, the at least one of palladium, platinum, ruthenium, or rhodium may be supported by activated carbon or silica. In some embodiments of the present invention, the metallic catalyst may include rhodium supported by activated carbon.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are incorporated into and form a part of the specification to illustrate examples of how the aspects, embodiments, or configurations can be made and used and are not to be construed as limiting the aspects, embodiments, or configurations to only the illustrated and described examples. Further features and advantages will become apparent from the following, more detailed description of the various aspects, embodiments, or configurations.

FIGS. 1 a-c illustrate biorefinery processes for the conversion of biomass to chemical intermediates and final products, according to exemplary embodiments of the present invention.

FIG. 2 illustrates engineered metabolic pathways in P. putida for converting both lignin depolymerization products and polysaccharide depolymerization products to muconic acid, according to exemplary embodiments of the present invention.

FIG. 3 summarizes engineered modifications to the genome of a P. putida strain to increase the production of muconic acid, followed by conversion to chemical intermediates, according to exemplary embodiments of the present invention.

FIG. 4 summarizes experimental culture results obtained using an engineered strain of Pseudomonas putida metabolizing various substrates for their conversion to muconic acid, according to exemplary embodiments of the present invention.

FIG. 5 summarizes experimental results from a DO-stat fed-batch experiment utilizing an engineered strain of P. putida (KT2440-CJ103) to convert p-coumarate to muconic acid, according to exemplary embodiments of the present invention.

FIG. 6 summarizes experimental results from a DO-stat fed-batch experiment utilizing an engineered strain of P. putida (KT2440-CJ103) to convert benzoate to muconic acid, according to exemplary embodiments of the present invention.

FIGS. 7 a-c summarize experimental results obtained from an engineered strain of P. putida that coexpresses decarboxylase subunits, EcdB and EcdD, to enhance the activity of the protocatechuate decarboxylase, AroY, according to exemplary embodiments of the present invention.

FIGS. 8 a-d summarize experimental results obtained from an engineered strain of P. putida that over-expresses VanAB for enhanced conversion of vanillate to protocatechuate to enable enhanced muconic acid production, according to exemplary embodiments of the present invention.

FIGS. 9 a-h summarize experimental results obtained from an engineered strain of P. putida with deregulation of Carbon Catabolite Repression to enhance aromatic catabolism and, subsequently, increase production of muconic acid from aromatic molecules, according to exemplary embodiments of the present invention.

FIGS. 10 a-b summarize experimental results obtained from an engineered strain of P. putida modified to express (−)-3-dehydroshikimate dehydratase, AsbF, the protocatechuate decarboxylase, AroY, and the protocatechuate subunit, EcdB, for enhanced production of muconic acid from sugars, according to exemplary embodiments of the present invention.

FIG. 11 tabulates primer sequences used to genetically engineer some of the P. putida strains engineered for improved muconic acid production, according to exemplary embodiments of the present invention. Depicted are LP29 (SEQ ID NO:39), LP30 (SEQ ID NO:40), LP31 (SEQ ID NO:41), LP32 (SEQ ID NO:42), LP33 (SEQ ID NO:43), LP34 (SEQ ID NO:44), LP48 (SEQ ID NO:45), LP49 (SEQ ID NO:46), LP50 (SEQ ID NO:47), LP51 (SEQ ID NO:48), LP53 (SEQ ID NO:49), LP54 (SEQ ID NO:50), oCJ100 (SEQ ID NO:51), oCJ101 (SEQ ID NO:52), oCJ102 (SEQ ID NO:53), oCJ103 (SEQ ID NO:54), oCJ165 (SEQ ID NO:55), and oCJ166 (SEQ ID NO:56).

FIGS. 12 a-c show experimental results comparing muconic acid production of a genetically engineered strain to the starting strain, according to exemplary embodiments of the present invention.

FIG. 13 illustrates post-biocatalysis steps to produce purified muconic acid and subsequently final produces, according to exemplary embodiments of the present invention.

FIG. 14 illustrates experimental results quantifying the components present in a culture broth before treatment with activated carbon, after treatment with activated carbon, and after crystallization, according to exemplary embodiments of the present invention.

FIG. 15 compares the elemental content of commercially available muconic acid biocatalysis-derived muconic acid at various purification/separation steps, according to exemplary embodiments of the present invention.

FIGS. 16 a-f illustrate purification of muconic acid biological culture media by activated carbon treatment, pH/temperature shift crystallization, and ethanol dissolution with microfiltration, according to exemplary embodiments of the present invention.

FIGS. 17 a-d summarize data from experiments evaluation catalytic hydrogenation of muconic acid to adipic acid, according to exemplary embodiments of the present invention.

FIG. 18 illustrates experimental data obtained screening catalysts for hydrogenating muconic acid to adipic acid using noble metal catalysts, according to exemplary embodiments of the present invention.

FIGS. 19 a-j summarize XRD spectra of virgin catalysts used for batch reactor screening experiments and after metal loading and catalyst reduction, according to exemplary embodiments of the present invention. Spectra were also provided for blank powdered silica and activated carbon supports for reference (a, b).

FIGS. 20 a-b illustrate muconic acid hydrogenation activity for platinum group metals (PGM) on powder activated carbon and silica supports in batch (a), and PGM leaching after 35 min exposure to reaction conditions (b), according to exemplary embodiments of the present invention.

FIGS. 21 a-j summarize data from batch reactor catalyst activity screening for muconic acid hydrogenation), according to exemplary embodiments of the present invention. Reactions were performed in a minimum of duplicate batch reactors, with error bars indicating standard deviations. Reaction conditions were as follows: temperature 24° C., muconic acid 200 mg, catalyst loading 15 mg, hydrogen pressure 24 bar, ethanol solvent 19.8 g.

FIG. 22 illustrates product molar yields, catalyst bed temperature, and liquid feed rate during the 100-h time-on-stream stability test of 1% Rh/AC for muconic acid hydrogenation, according to exemplary embodiments of the present invention. Reaction conditions were as follows: muconic acid 1 wt % in ethanol, temperature and liquid flow rate as indicated, hydrogen 200 sccm at 24 bar, catalyst loading 1100 mg.

FIG. 23 illustrates preliminary trickle bed reactor results with 1% Pd/AC granules, demonstrating ˜12 h to reach stead-state activity, according to exemplary embodiments of the present invention. Reaction conditions were as follows: muconic acid 1 wt % in ethanol, liquid flow rate 0.5 mL/min, catalyst bed temperature 72° C., hydrogen 200 sccm at 24 bar, and catalyst loading 200 mg of 1% Pd/AC granules.

FIG. 24 illustrates a polymerization scheme for reacting bio-adipic acid with 1,6-hexanediamine to produce Nylon-6,6 (left), according to exemplary embodiments of the present invention. This method using bio-adipic acid produced from the catalytic hydrogenation of muconic acid (right). Adipic acid was initially reacted with thionyl chloride and dissolved to cyclohexane, prior to adding the solution to a basic 1,6-hexanediamine aqueous solution. Nylon “rope” was then pulled from the biphasic solution interface for subsequent characterization.

FIG. 25 illustrates a flow diagram for the separation/purification and upgrading areas of a biorefinery for the conversion of lignocellulosic materials to muconic acid and subsequently to adipic acid, according to exemplary embodiments of the present invention.

REFERENCE NUMBERS

100 biorefinery 110 lignocellulosic biomass 120 pretreatment-fractionation 130 lignin depolymerization 135 polysaccharide depolymerization 140 microbial catalysis 150 separation/purification 160 catalytic hydrogenation 170 upgrading 200 bioreactor 210 muconic acid containing culture broth 220 purified muconic acid 230 final products

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description illustrates the invention by way of example and not by way of limitation. This description will clearly enable one skilled in the art to make and use the invention.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes”. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

References in the specification to “one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiment described may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases are not necessarily referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, are described with reference to a suitable host microorganism such as species from the Pseudomonas genus and their corresponding metabolic reactions or a suitable source microorganism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of microorganisms and the high level of skill in the area of genomics, those skilled in the art will readily be able to apply the teachings and guidance provided herein to essentially all other microorganisms. For example, the Pseudomonas metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Examples of other species include Sphingobium species (sp.) SYK-6, Rhodococcus jostii, Cupriavidus necator, Acinetobacter sp. ADP1, Amycolatopsis sp. ATCC 39116, E. coli, S. cerevisae, and/or fungi. Such genetic alterations include, for example, genetic alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene replacements.

Disclosed herein are methods for the integrated production of fuels, chemicals or materials from biomass, including lignin, cellulose, and hemicellulose, via catabolic pathways in bacteria. These methods enable a biological funneling approach for heterogeneous aromatic streams, thus opening a new route to produce renewable chemicals and fuels from biomass. Methods to couple this biological funneling to upstream lignin, cellulose, and/or hemicellulose depolymerization and downstream catalytic upgrading processes, thereby enabling a versatile, general approach to valorize lignin are also disclosed.

The methods presented herein may include the steps of lignin, cellulose, and/or hemicellulose depolymerization, biological funneling to a desired intermediate, followed by recovery and transformation to a value-added product. There is significant versatility in each step of this process such that it can be adapted to various feedstocks (e.g. raw materials), unit operations, and targeted fuel and chemical portfolios.

FIG. 1 a illustrates an exemplary biorefinery 100 that utilizes lignocellulosic biomass 110 to produce a high volume, commodity chemical, in this case adipic acid. FIG. 1 a illustrates corn and/or corn stover as the lignocellulosic biomass 100 raw material (the carbon, oxygen, and hydrogen source). However, other examples of lignocellulosic biomass 110 that may be processed by such a biorefinery 100 include wheat straw, bagasse, wood, wood chips, bark, grass, municipal solid waste, and any other common and/or high volume biomass source characterized by a high content of cellulose, hemicellulose, lignin, or combinations thereof. In some embodiments, the biomass utilized may include at least one of wood, wood chips, bark, sawdust, wood pellets, wood briquettes, forestry waste, pine, poplar, willow, Eucalyptus, Nothofagus, sycamore, ash, miscanthus, switchgrass, reed canary grass, rye, giant reed, hemp, bamboo, sugar cane, bagasse, corn, corn stover, wheat, wheat straw, sugar beets, sorghum, rapeseed, waste vegetable oil, palm oil, algae, municipal solid waste, and/or yard clippings.

The biomass feed stream may be provided by mechanical conveyor and/or pneumatically. One skilled in the art will recognize that some preprocessing of the biomass may be required to enable the providing step. Examples of preprocessing include size reduction, screening, filtering, washing, and combinations thereof. Size reduction may be accomplished by chopping, cutting, grinding, and combinations thereof, using for example, a hammer-mill and/or knife-mill.

After receiving the lignocellulosic biomass, FIG. 1 a illustrates that one or more pretreatment-fractionation operations 120 may pretreat and then separate the lignocellulosic biomass 110 into its component building blocks; e.g. lignin, cellulose, and hemicellulose. FIG. 1 a also illustrates that the polysaccharide components may be separated from a main processing stream, resulting in a lignin-rich stream. The polysaccharides may then be directed to separate and independent processing operations for conversion to, for example, sugars, chemicals, and/or fuels by dedicated processing operations (not shown). The lignin stream may be subsequently directed through various processing operations that may include lignin depolymerization operations 130 and microbial catalysis operations 140, resulting in the production of useful chemical intermediates, for example muconic acid. The intermediate chemicals produced may then be processed through one or more separation/purification operations 150 to yield purified intermediate components (e.g. muconic acid), after which the intermediates may be packaged and/or stored for delivery and sale, or directed to down-stream processing, for subsequent upgrading to higher value chemicals and/or fuels. FIG. 1 a illustrates an exemplary upgrading operation that includes catalytic hydrogenation 160, to convert muconic acid to adipic acid. Thus, biological funneling of lignin-derived monomers by engineered microorganisms may be combined with downstream upgrading to facilitate the development of an immense range of products.

Any pretreatment, fractionation, or depolymerization method that generates a lignin-containing stream is suitable for use in the methods described herein. Referring again to FIG. 1 a, pretreatment and/or fractionation operations 120 of a lignocellulosic biomass 110 such as corn stover may include contacting the lignocellulosic biomass with an alkaline compound or biological or chemical catalyst to generate a soluble lignin-containing stream. Exemplary processing conditions include treatment with a base such as sodium hydroxide for a time ranging from several minutes to several hours at a temperature ranging from about 50° C. to about 200° C. at a solids loading of about 5 wt % to about 10 wt % solids in a mixer. These conditions may be varied by one skilled in the art, commensurate with the biomass source, the chemical or biological catalyst used, and other process and physical property parameters. Some embodiments of the present invention include lignin-polysaccharides separations using separation methods known to one skilled in the art. Some examples of lignin-polysaccharide separation methods which may be used in the present invention are described in U.S. Pat. Nos. 5,730,837; 2,037,001; 1,594,389; 1,888,025; 2,042,705; 3,932,207; 4,520,105; and 4,594,130, all of which are incorporated herein by reference in their entirety. Should definition discrepancies arise between publications incorporated herein by reference and the present written description and claims, the definitions provided herein shall over rule any other external definitions.

Referring again to FIG. 1 a, lignin depolymerization operations 130 to low molecular weight aromatics may be achieved via thermal, biological, and/or catalytic processing. For example, white rot fungi and some bacteria may depolymerize lignin to its monomeric constituents using powerful oxidative enzymes. Lignin is composed of three monomeric phenylpropanoid units that differ in their degree of methoxylation, which are polymerized by carbon-carbon and carbon-oxygen bonds formed during lignin biosynthesis via radical coupling reactions. Thus, lignin depolymerization operations 130 may include a broad range of catalytic, thermal, and biological routes, and may yield a chemically heterogeneous pool of depolymerization intermediates, which may include for example, p-coumaric acid, ferulic acid, benzoic acid, phenol, coniferyl alcohol, caffeic acid, vanillin, and/or 4-hydroxybenzoic acid.

Referring again to FIG. 1 a, microbial catalysis 140 may include biological funneling of one or more lignin depolymerization products/intermediates to produce molecules from acetyl-CoA, the tricarboxylic acid cycle, and beyond in carbon metabolism. In aerobic systems, a microbial catalysis operation 140 may utilize aromatic-catabolizing microorganisms that involve the use of “upper pathways”. These “upper pathways” may employ a diverse battery of enzymes to funnel aromatic monomers and oligomers to key central intermediates, such as catechol and protocatechuic acid. From these central intermediates, dioxygenase enzymes may oxidatively cleave carbon-carbon bonds in the aromatic rings to produce ring-opened species that may then be “funneled” to central carbon metabolism routes, which may then ultimately lead to the tricarboxylic acid cycle. These catabolic pathways may enable engineered microorganisms to utilize a broad range of both natural and xenobiotic aromatic molecules as carbon and energy sources. In terms of chemicals and/or biofuels production, these metabolic pathways offer a direct, powerful means to biologically ‘funnel’ the heterogeneous slate of molecules produced from lignin depolymerization into either fuels or chemicals. An example of a useful intermediate chemical resulting from a microbial catalysis operation 140 (via biological funneling), as described above, is muconic acid.

The separation/purification operations 150 of the fuels, chemicals, and/or intermediates resulting from the microbial catalysis operation 140 may include a variety of unit operations. The selection of one or more specific unit operations for the separation/purification operation 150 will depend on the on the design criteria and operating conditions of the upstream processing; e.g. type of lignocellulosic biomass 110 utilized, and pretreatment-fractionation operation 120 efficiency and yield, and the species targeted for removal from the lignin stream. The separation/purification operation 150 needed may also depend on the details of the microbial catalysis operation 140; e.g. type of microorganism used, culture broth composition, etc. However, examples of unit operations for the separation/purification operation 150 may include filtration, centrifugation, distillation, vacuum distillation, adsorption, membrane separations, cross-flow membrane filtration, crystallization, and/or any other suitable separation/purification unit operations. For example, culture broth containing muconic acid from microbial catalysis 140 may be centrifuged and/or filtered to produce a solids stream (e.g. liquid stream with a relatively high concentration of cell matter) and a substantially solids-free liquid stream containing muconic acid. The muconic acid containing stream may then be treated by one or more unit operations, e.g. adsorption, crystallization, to produce a relatively pure muconic acid stream capable of downstream upgrading to value-added final products (e.g. chemicals, fuels, etc.)

Alternatively, a biorefinery 100 may also be designed to utilize predominantly the cellulosic components of the lignocellulosic biomass 110 to produce useful fuels and chemicals, as illustrated in FIG. 1 b. In this example, lignin may be separated from the polysaccharides to form a polysaccharide-rich stream. The lignin may then be directed to separate and independent processing operations for conversion to lignin depolymerization products, for example aromatic compounds. The polysaccharide stream may be subsequently directed through various processing operations that may include polysaccharide depolymerization operations 135 and microbial catalysis operations 140, resulting in the production of useful chemical intermediates, for example muconic acid. The microbes (microorganisms) utilized for the microbial catalysis operation 140 may be engineered in this example to funnel polysaccharide degradation products (e.g. glucose, xylose, arabinose) to protocatechuic acid and/or catechol and subsequently to muconic acid. The muconic acid containing stream from the microbial catalysis operation 140 of FIG. 1 b, may then be subsequently separated and/or purified in a separation/purification operation 150 operations. These operations for polysaccharide-based products may be significantly different from the separation/purification operations 150 utilized for the processing of the lignin-based products of FIG. 1 a, because the microorganisms engineered to metabolize lignin depolymerization products may be significantly different from the microorganisms engineered to metabolize polysaccharide depolymerization products, as may be the culture media/broth and culture conditions. Thus, another biorefinery example may begin with something like the biorefinery of FIG. 1 a, where the polysaccharide stream removed by the pretreatment-fractionation 120 operations may be fed to a polysaccharide depolymerization operation 130 operation like that of FIG. 1 b. In so doing, both the polysaccharide stream and the lignin stream may have their own dedicated depolymerization operation, microbial catalysis operation, separation/purification operation, and upgrading operation (e.g. catalytic hydrogenation).

Alternatively, one or more microorganisms may be engineered to process both lignin depolymerization products and polysaccharide depolymerization products in a single microbial catalysis step. This option is illustrated in FIG. 1 c. Thus, FIG. 1 c combines FIG. 1 a and FIG. 1 b into a single series of operations wherein removal of a constituent biomass component (e.g. lignin or polysaccharides) does not occur. Instead, any pretreatment-fractionation steps (not shown) may be limited to, for example, washing steps for removing debris, metal removal steps, etc. Thus, biomass depolymerization 137 of lignin and cellulose/hemicellulose may occur simultaneously in a single processing step. Similarly, microbial catalysis 140 of the resultant lignin and polysaccharides depolymerization products may occur simultaneously in a single processing step (e.g. in a single bioreactor). Simultaneous culturing of the lignin and polysaccharides depolymerization products may include one biocatalyst/microorganism that has been engineered to process both lignin and polysaccharide depolymerization products to produce useful products (e.g. muconic acid). Alternatively, one or more biocatalysts/organisms may be engineered and/or optimized for the conversion of lignin depolymerization products to useful chemical intermediates and one or more additional biocatalysts/microorganisms may be engineered and/or optimized for the conversion of polysaccharides to useful chemical intermediates, with the various microorganisms used together in one or more culturing steps. Once the microbial catalysis 140 produces muconic acid, the resultant culture broth containing the muconic acid will be treated in a separations/purification operation 150 to produce a muconic acid stream of sufficient purity and quality to allow subsequent upgrading 170 (e.g. hydrogenation) to final products; e.g. adipic acid and/or nylon-6,6.

This disclosure will focus next on details and examples regarding the microbial catalysis 140 portion of the biorefinery 100 and will return later to the separation/purification operation 150 and the upgrading 170 portions of the biorefinery 100.

Microbial Catalysis

Microorganisms engineered and/or modified to funnel lignin depolymerization products and/or polysaccharide depolymerization products to useful molecules (e.g. chemicals and/or fuels) may include prokaryotes such as bacteria or eukaryotes such as yeasts or fungi. Further examples include Pseudomonas sp., Sphingobium sp. SYK-6, Rhodococcus jostii, Cupriavidus necator, Acinetobacter sp. ADP1, Amycolatopsis sp. ATCC 39116, E. coli, S. cerevisae, bacterial species from the genera Streptomyces or Bacillus, and/or fungi. Another example of a genetically modified bacterium may include the genus Pseudomonas. In some cases, the genetically modified microorganism may include at least one Pseudomonas species such as aeruginosa, chlororaphis, fluorescens, pertucinogena, putida, stutzeri, syringae, and/or incertai sedis. In other cases, the genetically modified microorganism may include at least one of P. putida, P. fluorescens, and/or P. stutzeri. In still other cases, the genetically modified microorganism may include at least one strain of Pseudomonas putida KT2440.

In some embodiments, the genetically modified microorganism may include at least one of P. putida group, including at least one of P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva, P. plecoglossicida, and/or P. putida. In some embodiments, the genetically modified microorganism may include at least one of the P. fluorescens group, including at least one of P. antarctica, P. azotoformans, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridian, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, and/or P. tolaasii.

As used herein, “exogenous” refers to something originating from another genetic source or species. So, as used herein, a genetically modified bacterium refers to a bacterium wherein the genetic modification is either the addition of genetic material from another species and/or the deletion of a portion of its own endogenous genetic material. So, as used herein, “endogenous” refers to native or naturally occurring genetic material of the microorganism itself.

As used herein, “gene” and “genetic source” and “genetic material” refer to a segment of nucleic acid that encodes an individual protein or RNA molecule (also referred to as a “coding sequence” or “coding region”) and may include non-coding regions (“introns”) and/or associated regulatory regions such as promoters, operators, terminators and the like, that may be located upstream or downstream of the coding sequence.

Genetic modifications and/or engineering to a microorganism to enable and/or improve the funneling of lignin depolymerization products and/or polysaccharide depolymerization products to useful intermediate compounds and/or useful fuels and/or chemicals may include at least one exogenous gene addition, at least one endogenous gene deletion, and/or the over-expression of at least one endogenous and/or exogenous gene. Such additions may be genomic and/or include the addition of plasmids that contain the desired gene.

FIG. 2 illustrates exemplary engineered pathways for enabling an engineered microorganism to metabolize at least one of lignin, cellulose, hemicellulose, lignin depolymerization products, cellulose depolymerization products, and/or hemicellulose depolymerization products, to produce muconic acid. In this example, a strain of P. putida may include pathways for metabolizing lignin depolymerization products (e.g. aromatic compounds) and for metabolizing polysaccharide depolymerization products (e.g. glucose, xylose, arabinose, mannose, galactose, and/or rhamnose) to maximize the production of muconic acid. Referring again to FIG. 2, the exemplary genetically modified microorganism may convert coniferyl alcohol to coniferyl aldehyde and/or the genetically modified microorganism may convert the coniferyl aldehyde to ferulic acid and/or the genetically modified microorganism may convert the ferulic acid to at least one of vanillin and/or vanillic acid. A genetically modified microorganism may convert the vanillin to vanillic acid and/or the genetically modified microorganism may convert the vanillic acid to protocatechuic acid. A genetically modified microorganism may convert p-coumaric acid to 4-hydroxybenzoic acid and/or the genetically modified microorganism may convert 4-hydroxybenzoic acid to protocatechuic acid. A genetically modified microorganism may convert at least one of glucose, xylose, and/or arabinose to 3-dehydroshikimate (DHS) and/or the genetically modified microorganism may convert DHS to protocatechuic acid. A genetically modified microorganism may convert protocatechuic acid to catechol. A genetically modified microorganism may convert phenol to catechol. A genetically modified microorganism may convert benzoic acid to benzoic acid diol. A genetically modified microorganism may convert benzoic acid diol to catechol. A genetically modified microorganism may convert the catechol to muconic acid. In some examples, a genetically modified microorganism may be engineered to complete one or more of the reactions described above.

FIG. 2 illustrates that various genes coding a number of different enzymes may be manipulated, for example removed, added, over-expressed, and/or under-expressed to genetically engineer a microorganism (e.g. P. putida) to maximize the yield and/or selectivity of the target product molecule (e.g. muconic acid). For example, a microorganism may be engineered such that one or exogenous genes may be added to the microorganism. For example, a microorganism may be engineered such that one or more genes encoding exogenous decarboxylases may be added to the microorganism. A microorganism may be engineered such that one or more genes encoding exogenous monooxygenases may be added to the microorganism. A microorganism may be engineered such that one or more exogenous genes encoding dehydratases may be added to the microorganism. A microorganism may be engineered such that one or more genes encoding one or more exogenous monooxygenases, decarboxylases, and/or dehydratases may be added to the microorganism. Gene additions may be genomic additions or through the use of plasmids. Genes may be provided by any suitable and compatible microorganism for example Enterobacter cloacae, Pseudomonas sp., and/or Bacillus cereus. A microorganism may be engineered such that one or more genes encoding endogenous demethylases may be over-expressed by the microorganism.

In some embodiments, a microorganism may be engineered for optimized muconic acid production such that one or more endogenous genes may be removed from the microorganism. For example, a microorganism may be engineered such that one or more genes encoding catabolite repression control proteins may be removed, for example the gene encoding the Crc protein (see SEQ ID NO:13 and SEQ ID NO:14). A microorganism may be engineered such that one or more endogenous genes encoding dioxygenases may be removed from the microorganism. A microorganism may be engineered such that one or more endogenous genes encoding muconating lactonizing enzymes may be removed from the microorganism. A microorganism may be engineered such that one or more endogenous genes encoding muconolactone isomerases may be removed from the microorganism. In some embodiments, a microorganism may be engineered such that at least one gene encoding a catabolite repression control protein, a dioxygenase, a muconating lactonizing enzyme, and/or a muconolactone isomerase are removed from the microorganism.

A microorganism may be optimized for muconic acid production by the addition of several genes. For example, an exogenous decarboxylase that may be added to a microorganism may include 3,4-dihydroxybenzoate decarboxylase from Enterobacter cloacae subsp. cloacae (ATCC 13047). Such an exogenous decarboxylase may be encoded by an aroY gene (see SEQ ID NO:17 for the DNA sequence with the corresponding amino acid sequence shown by SEQ ID NO:18). In some embodiments, an exogenous dehydratase added to a microorganism may be from at least one of Klebsiella pneumoniae, K. oxytoca, K. planticola, K. ornithinolytica, K. terrigena, Enterobacter cloacae, Enterobacter cancerogenus, Enterobacter hormaechei, Enterobacter mori or combinations thereof. In some embodiments, an exogenous dehydratase may be engineered into a microorganism, where the exogenous dehydratase may be encoded by an aroZ gene. A microorganism may be modified by the addition of at least one exogenous monooxygenase encoded by at least one gene of dmpK (see SEQ ID NO:23 and SEQ ID NO:24), dmpL (see SEQ ID NO:25 and SEQ ID NO:26), dmpM (see SEQ ID NO:27 and SEQ ID NO:28), dmpN (see SEQ ID NO:29 and SEQ ID NO:30), dmpO (see SEQ ID NO:31 and SEQ ID NO:32), and/or dmpP (see SEQ ID NO:33 and SEQ ID NO:34) from the microorganism Pseudomonas sp. CF600 or the gene pheA from Pseudomonas sp. EST1001 (SEQ ID NO:35 and SEQ ID NO:36). For example, a microorganism may be modified to include the addition of each of dmpK, dmpL, dmpM, dmpN, dmpO, and dmpP, where such a modification is referred to herein as the addition of dmpKLMNOP. In some embodiments, at least one exogenous decarboxylase may be engineered into a microorganism, where the at least one exogenous decarboxylase may further include at least one gene of ecdB (see SEQ ID NO:19 and SEQ ID NO:20) and/or ecdD (see SEQ ID NO:21 and SEQ ID NO:22). At least one exogenous dehydratase may be engineered into a microorganism, where the exogenous dehydratase may be encoded by asbF (see SEQ ID NO:15 and SEQ ID NO:16). At least one endogenous demethylase may be over-expressed in an microorganism, where the demethylase may be encoded by at least one of vanA (see SEQ ID NO:5 and SEQ ID NO:6), vanB (see SEQ ID NO:7 and SEQ ID NO:8), or ligM (SEQ ID NO:37 and SEQ ID NO:38).

A microorganism may be optimized for muconic acid production by the deletion of at least one gene encoding a muconate lactonizing enzyme and/or a muconolactone isomerase, such as catB (see SEQ ID NO:1 and SEQ ID NO:2) and/or catC (see SEQ ID NO:3 and SEQ ID NO:4). In some examples, a microorganism may be manipulated to maximize muconic acid production by the removal of at least one dioxygenase, where the dioxygenase may be encoded by at least one gene of pcaH (see SEQ ID NO:9 and SEQ ID NO:10) and/or pcaG (see SEQ ID NO:11 and SEQ ID NO:12).

In some examples, at least one endogenous gene deletion to modify a microorganism for improved muconic acid production may include the deletion of at least one gene that encodes at least one enzyme that metabolizes muconic acid to a different molecule. In some embodiments, at least one endogenous gene deletion from a microorganism may include the deletion of at least one gene that encodes at least one enzyme in the β-ketoadipate pathway that metabolizes muconic acid to a different molecule.

In still further embodiments, the modified (e.g. engineered) microorganism may include at least one exogenous gene addition that encodes at least one enzyme of the pentose phosphate pathway and the addition of at least one gene that encodes at least one enzyme of a glycolytic pathway, such as the Embden-Meyerhof-Parnas pathway or the Entner-Doudoroff pathway. For example, some of the exogenous genes that may be added to a microorganism for improved muconic acid production may encode enzymes from the pentose phosphate pathway, such as at least one of glucose-6-phosphate dehydrogenase, gluconolactonase, 6-phosphogluconate dehydrogenase, ribulose-5-phosphate isomerase, ribulose-5-phosphate-3-epimerase, transketolase, and/or transaldolase. As another example, some of the exogenous genes that may be added to a microorganism for improved muconic acid production may encode enzymes from the Glycolysis pathway, such as at least one of hexokinase, phosphofructokinase, fructose-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoclycerate kinase, phosphoglycerate mutase, enolase, and/or pyruvate kinase. In further embodiments, the modified microorganism may include at least one endogenous gene deletion where at the gene deleted encodes an enzyme for converting 3-dehydroshikimate (DHS) to an amino acid.

As used herein, the term “homologous” sequences of nucleic acids and proteins refer to sequences that have a statistically significant degree of similarity. In some embodiments of the present invention, any of the genes and the proteins and/or enzymes that they encode, e.g. dehydratases, decarboxylases, dioxygenases, monooxygenases, genes and enzymes from the pentose phosphate pathway, genes and enzymes from the Glycolysis pathway, may include nucleic acid and/or amino acid sequences that are homologous to the specific examples given in that the homologs have nucleic acid and/or amino acid sequences that are at least 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to or identical to the sequences of the exemplary enzymes provided above. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is to be understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that each encode substantially the same protein.

As a result of at least one of the genetic modifications described above, a genetically engineered microorganism may be used to metabolize at least one of lignin, cellulose, hemicellose, or combinations thereof, to produce useful final products, chemicals, and chemical intermediates. Further, a modified microorganism may be used to metabolize at least one of the intermediations of lignin depolymerization, cellulose depolymerization, hemicellose depolymerization, or combinations thereof, to produce useful final products, chemicals, and chemical intermediates. For example, a modified microorganism (e.g. a bacterial strain such as P. putida) may be engineered to metabolize at least one of carbohydrates, cellobiose, polysaccharides, C5 sugars, C6 sugars, and/or lignin depolymerization products. Sugars that may be metabolized in some embodiments by modified microorganisms described herein may include at least one of xylose, glucose, galactose, arabinose, mannose, or combinations thereof. Lignin depolymerization products that may be metabolized by some embodiments of the modified microorganisms described herein may include least one of phenylpropanoid units, coniferyl alcohol-derived constituents, syringyl structures, coniferyl structures, p-coumaryl groups, or mixtures thereof. In still further embodiments, compounds that may be metabolized by some examples of the modified microorganisms as described herein may include at least one of p-coumaryl alcohol, syringyl alcohol, coniferyl alcohol, coniferyl aldehyde, ferulic acid, feruloyl-CoA, vanillin, vanillic acid, caffeic acid, or mixtures thereof. Other chemical compounds that may be metabolized by some examples of the genetically engineered microorganisms described herein include at least one of 3-dehydroshikimate, p-coumeric acid, 4-hydroxybenzoic acid, phenol, benzoic acid, benzoic acid diol, or mixtures thereof. In some embodiments, a modified microorganism as described herein, engineered to metabolize at least one of lignin, cellulose, hemicellulose, and their respective depolymerization products, may achieve this metabolism by use of at least one of the β-ketoadipate pathway, the Embden-Meyerhof-Parnas pathway, the Entner-Doudoroff pathway, the pentose phosphate pathway, and/or an amino acid synthesis pathway.

Referring again to FIG. 2, several organic acids are shown: e.g. ferulic acid, vanillic acid, caffeic acid, protocatechuic acid, p-coumaric acid, 4-hydroxybenzoic acid, benzoic acid, etc. Organic acids in aqueous solutions will reach an equilibrium concentration/ratio of the conjugate acid (e.g., muconic acid) with the conjugate base (e.g., muconate). The relationships defining these equilibrium concentrations and ratios are controlled at least by the acid dissociation constant and solution pH and often both the acid and base form will be present in an aqueous solution (e.g. culture broth). Thus, for simplification purposes, as used herein reference to either the conjugate acid form (e.g., muconic acid) or the conjugate base form (e.g., muconate) of a molecule means both forms of molecule are present unless specified otherwise.

FIG. 4 summarizes experimental results from an engineered P. putida KT2440 strain. For this example, the strain was modified to capture aromatic species metabolized through protocatechuic acid. This was accomplished by replacing the gene pcaHG encoding a protocatechuate 3,4 dioxygenase with aroY encoding a protocatechuate decarboxylase from Enterobacter cloacae. This enabled the conversion of protocatechuic acid and upstream metabolites to catechol while simultaneously eliminating further catabolism of protocatechuic acid to β-ketoadipate via the TCA-cycle (see FIG. 2, β-ketoadipate not shown). The same P. putida strain was then further engineered to expand substrate utilization and to eliminate further metabolism of muconic acid. This was accomplished be removing CatA and CatB by removing a genomic section containing catR, catBC, and the promoter for catBCA and replacing it with the Ptac promoter, which allowed the constitutive expression of catA. Lastly, as phenol is a commonly derived lignin intermediate, the genes encoding the phenol monooxygenase from Pseudomonas sp. CF600, dmpKLMNOP were integrated into the genome downstream of catA to form an operon driven by the Ptac promoter (see FIG. 3).

The metabolic performance of the engineered P. putida strain (called KT2440-CJ103) was then evaluated in shake-flask experiments to demonstrate substrate utilization and production of muconic acid from model lignin-derived monomers, using acetate as a carbon and energy source. FIG. 4 illustrates the metabolism of benzoate (benzoic acid), p-coumarate, phenol, 4-hydroxybenzoic acid (4-HBA), ferulate (ferulic acid), protocatechuate (protocatechuic acid), vanillin, caffeate (caffeic acid), coniferyl alcohol, and catechol by KT2440-CJ103. KT2440-CJ103 successfully produced muconic acid from catechol, phenol, and benzoate via the catechol branch (see FIG. 2), as well as from protocatechuate, coniferyl alcohol, ferulate, vanillin, caffeate, p-coumarate, and 4-hydroxybenzoate via the protocatechuate branch. Muconic acid yields ranged from about 14% using coniferyl alcohol as a substrate to about 93% utilizing benzoate. Compounds metabolized through vanillate (see FIG. 2; e.g. coniferyl alcohol, ferulate, and vanillin) demonstrated lower yields with accumulation of the intermediate vanillate. In contrast, compounds metabolized through the catechol branch (see FIG. 2; e.g. phenol, catechol, benzoate), as well as p-coumarate and 4-HBA, provided higher yields.

The performance of the engineered P. putida strain (KT2440-CJ103) was also studied in a fed-batch bioreactor experiment to understand the effects increased aeration, pH control, and a metered dosing of substrates on P. putida growth and the conversion of substrate to muconic acid. The results are illustrated in FIG. 5. Dissolved oxygen static (DO-stat) fed-batch biological conversion by KT2440-CJ103 yielded a muconic acid titer of about 13.5 g/L after about 78.5 hours using p-coumarate as a model lignin monomer substrate, over 15 times greater than the shake flask results shown in FIG. 4. Preliminary experiments indicated that muconic acid production from p-coumarate was significantly inhibited by the presence of excess glucose or acetate, potentially due to catabolite repression control or other regulatory inhibition. Therefore, DO-stat was used to maintain glucose levels below 1 mM while co-feeding p-coumarate and ammonium sulfate. During the course of cultivation, protocatechuate (protocatechuic acid) buildup occurred. Moreover, as the cultivation progressed past 60 hours, muconic acid concentrations plateaued and 4-HBA, a metabolite upstream of protocatechuic acid, accumulated.

FIG. 6 illustrates another set of fed-batch results where the P. putida KT2440-CJ103 strain was used to convert benzoate (benzoic acid) to muconic acid. In this example, glucose was used as a carbon source to support biological growth and function. Benzoate feeding was based on DO stat control, while pH was controlled using NaOH, resulting in sodium muconate as the predominant species at a pH of about 7. Muconate was produced at a titer of about 7.97 g/L after about 32 hours, while residual benzoic acid was present at about 0.94 g/L. After terminating the fed-batch run, cells were removed by centrifugation and filtration for subsequent broth processing.

Numerous other strains of P. putida KT2440 have been engineered and tested for their ability to convert lignin depolymerization products to muconic acid, several examples of which follow below.

Example 1

Co-expression of decarboxylase subunits, EcdB and EcdD, to enhance the activity of the protocatechuate decarboxylase, AroY and, subsequently, increase production of muconic acid from aromatic molecules metabolized through protocatechuate.

As mentioned above, some experiments with P. putida KT2440-CJ103 for producing muconic acid from aromatic molecules metabolized through protocatechuate (PCA), including p-coumarate, 4-hydroxybenaote (4-HBA), ferulate, and vanillin, demonstrated an accumulation of protocatechuate that reduced muconic acid yields. This suggested that the activity of the heterologously expressed protocatechuate decarboxylase that converts protocatechuate to catechol, AroY, may be insufficient. In an attempt to eliminate this bottleneck, enzymes from Enterobacter cloacae subsp. cloacae (ATCC 13047), EcdB and EcdD along with AroY (also from Enterobacter cloacae subsp. cloacae (ATCC 13047)) were engineered into a P. putida strain that was otherwise engineered to produce muconic acid from aromatic molecules. Metabolism of p-coumarate with the co-expression of AroY with EcdB (FIG. 7 b) or with EcdB and EcdD (FIG. 7 c), exhibited increased activity relative to AroY alone (FIG. 7 a), which ultimately resulted in reduced accumulation of protocatechuate and greater production of muconic acid from p-coumarate.

Referring to FIG. 7 a which illustrates the performance of P. putida strain KT2440-CJ102, the text in parenthesis, ΔcatRBCA::Ptac:catA ΔpcaHG::Ptac:aroY, means the following. First, the gene or genes immediately following a Δ symbol have been deleted from the genome. The double-colon following the deleted gene(s) refers to replacing the deleted gene(s) with the genetic element, gene or genes that immediately follow the double-colon. Finally, the single colon refers to genetic fusion of the gene before the colon to the gene following the colon. Thus, two genetic modifications were made to strain KT2440 to produce new strain KT2440-CJ102 (see FIG. 7 a). First, the catRBCA genes were deleted and replaced by a DNA fragment comprising the Ptac promoter upstream of the catA gene and second, the pcaHG genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of the and aroY gene. Referring to FIG. 7 b, the KT2440-CJ183 strain was constructed by modifying the KT2440 strain: first, the catRBCA genes were deleted and replaced by a DNA fragment comprising the Ptac promoter upstream of catA and second, the pcaHG genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of aroY and ecdB. Referring to FIG. 7 c, the KT2440-CJ184 strain was constructed by modifying the KT2440 strain: first, the catRBCA genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of catA and second, the pcaHG genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of aroY, and ecdBD (ecdB and ecdD).

Example 2

Over-expression of VanAB for enhanced conversion of vanillate (aromatic pathways) to protocatechuate and, subsequently, increased production of muconic acid from coniferyl alcohol pathway metabolites.

In addition to the AroY “bottleneck” described above, a considerable accumulation of vanillate was observed in the P. putida KT1440-CJ103 when metabolites from the coniferyl alcohol degradation pathway including coniferyl alcohol, ferulate, and vanillin were used as substrates for the production of muconic acid. This resulted in a reduction in the amount of muconic acid produced. FIGS. 8 b-d illustrate the performance results of strains successfully engineered to remove this bottleneck, as demonstrated by reduced accumulation of vanillate and, thus, increased production of muconic acid relative to the empty vector control strain (FIG. 8 a). A P. putida strain with increased expression of VanA is shown in FIG. 8 b, a P. putida strain with increased expression of VanB is shown in FIG. 8 c), and a third P. putida strain with increased expression of both VanA and VanB is shown in FIG. 8 d. Unlike, the strains described above which involve genomic gene additions, VanA and VanB were added to the P. putida KT1440 strain by cloning the genes that encode these proteins into a broad-host range plasmid, pBTL-2, and transforming the resulting plasmids into P. putida KT2440-CJ102, where expression of these genes were driven constitutively by the Plac promoter in pBTL-2.

Example 3

Deregulation of Carbon Catabolite Repression to enhance aromatic catabolism and, subsequently, increase production of muconic acid from aromatic molecules.

In Pseudomonads such as P. putida KT2440, the Catabolite Repression Control (Crc) protein binds targeted RNAs encoding proteins involved in catabolism and, thereby, may inhibit their translation and, thus, their activity. Pathways that enable catabolism of less preferred substrates are inhibited by Crc until preferred substrates, those which provide more carbon and/or energy, have been depleted. Among the targets of Crc regulation is catabolism of aromatic molecules. As shown in FIGS. 9 a-h, deletion of Crc from P. putida KT2440-CJ102, resulted in less accumulation of intermediates from aromatic degradation, including 4-hydroxybenzoate and vanillate, which in turn resulted in higher production of muconic acid (see FIGS. 9 b, 9 d, 9 f, and 9 h), relative to the equivalent strain expressing Crc (see FIGS. 9 a, 9 c, 9 e, and 9 g). Thus, deletion of Crc represents a novel way to enhance the catabolism of aromatic molecules derived from lignin depolymerization and the subsequent production of molecules derived from them, such as muconic acid.

Example 4

Expression of (−)-3-dehydroshikimate dehydratase, AsbF, and the protocatechuate decarboxylase, AroY, for production of muconic acid from sugars.

Heterologous expression of a (−)-3-dehydroshikimate (3-DHS) dehydratase, a protocatechuate decarboxylase, and a catechol dioxygenase may convert 3-DHS, an intermediate in the biosynthesis of aromatic amino acids, to protocatechuate, which may then be converted to catechol and cleaved to form muconic acid. FIGS. 10 a-b demonstrate the production of muconic acid from glucose (FIG. 10 a) by the engineered P. putida strain KT2440-CJ156 and KT2440-CJ200.

Referring to FIG. 10 a, two genetic modifications were made to P. putida strain KT2440 to produce new P. putida strain KT2440-CJ156. First, the catRBCA genes were deleted and replaced by a DNA fragment comprising the Ptac promoter upstream of catA and second, the pcaHG genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of aroY from Enterobacter cloacae subsp. cloacae (ATCC 13047) and asbF from Bacillus cereus (ATCC 14579). Referring to FIG. 10 b, two genetic modifications were made to P. putida strain KT2440 to produce new P. putida strain KT2440-CJ200. First, the catRBCA genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of catA and second, the pcaHG genes were deleted and replaced with a DNA fragment comprising the Ptac promoter upstream of aroY from Enterobacter cloacae subsp. cloacae (ATCC 13047), ecdB from Enterobacter cloacae subsp. cloacae (ATCC 13047), and asbF from Bacillus cereus (ATCC 14579). Such engineered P. putida KT2440 strains have several advantages over less stress-tolerant, plasmid-bearing hosts such as E. coli or Saccharomyces cerevisiae. In addition, expression of ecdB more than doubled the amount of muconic acid produced from glucose (see FIG. 10 b), by increasing the activity of AroY. This pathway may be used to produce muconic acid from cellulose or hemicellulose-derived sugars such as glucose or xylose (or, for example, arabinose, mannose, galactose or rhamnose) as well as other bio-mass relevant feedstocks including, but not limited to acetate, and glycerol.

Example 5

Strains, plasmid construction, and gene replacement methods. The example presented here illustrates the methods used to genetically modify P. putida KT2440 to construct the various modified P. putida strains described above.

Competent NEB (New England Biolabs, Inc., Ipswich, Mass.) C2925 and Life Technologies (Grand Island, N.Y.) TOP10 was used for plasmid construction of cis,cis-muconate (muconic acid) producing and phenol utilizing strains, respectively. NEB 5-alpha F′I^(q) E. coli was used for all remaining plasmid constructions and were grown shaking at 225 rpm, 37° C., in LB Broth (Lennox) containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl or on LB plates containing 15 g/L agar, with either 10 μg/mL tetracycline or 50 μg/mL kanamycin. E. coli was transformed according to the manufacturer's instructions.

Q5® Hot Start High-Fidelity 2× Master Mix (NEB) and primers synthesized by IDT (Integrated DNA Technologies, Inc., Iowa) were used in all PCR amplification for plasmid construction. Primer sequences are shown in FIG. 11. Plasmids were assembled using Gibson Assembly® Master Mix (NEB) according to the manufacturer's instructions. The sequences of all plasmid inserts were confirmed using Sanger sequencing

Plasmids for gene replacement were constructed in pCM433 (Addgene Inc., Cambridge, Mass.)1 or pKl8mobsacB from ATCC (American Type Culture Collection, Manassas, Va.), both of which are unable to replicate in P. putida and contain antibiotic resistance genes to select for integration of the plasmid into the genome by homologous recombination and sacB to counterselect for recombination of the plasmid out of the genome. Plasmids for expression of vanA and/or vanB were constructed in pBTL-2 (Addgene Inc., Cambridge, Mass.), which is able to replicate in P. putida.

The pCM433-based integration vector used to replace catRBCA with Ptac:catA (pMFL22) was constructed by Gibson assembly of three PCR products: LP29 and LP33 were used to amplify the targeting region upstream from catA, LP30 and LP31 were used to amplify the Ptac promoter from Sigma pFLAG-CTC, LP32 and LP34 were used to amplify the entire coding region of catA including its native RBS. After assembly, the 2.2 kb fragment was amplified by PCR using primers LP29 and LP34, and cloned into the pCM433 vector using NotI sites.

The pK18mobsacB-based plasmid for integration of the phenol monooxygenase genes (pMFL56) was constructed by Gibson assembly of three PCR fragments using primers LP53 and LP48 to amplify the catA targeting region, LP49 and LP50 for amplification of six phenol monooxygenase genes, dmpKLMNOP using pVI1261 as the template (provided by Dr. Victoria Shingler from the Department of Molecular Biology at timed University), and primers LP51 and LP54 for amplification of the targeting region downstream from catA. Fragments were then cloned into pK18-mob vector using NotI sites.

In the plasmid for replacement of pcaHG with Ptac:aroY (pCJ023), the aroY gene (ADF69416) from Enterobacter cloacae ATCC13047 was optimized for expression in P. putida KT2440 using DNA 2.0's Gene Designer software and synthesized in two overlapping DNA fragments by IDT. The first fragment also contained the Ptac promoter, which was separated from the initiating ATG by a ribosome binding site with the sequence AGAGGAGGGAGA. These fragments were then assembled by Gibson assembly and Ptac:aroY was amplified from this assembly with primers oCJ165 and oCJ166. Approximately 1 kb regions upstream and downstream of pcaHG were amplified using oCJ100/oCJ101, and oCJ102/oCJ103, respectively. The upstream targeting region, Ptac:aroY, and the downstream targeting region were then assembled into pCM433 linearized with restriction enzymes AatII and Sad (NEB).

Gene replacement plasmids were transformed into P. putida strains by electroporation. LB broth was inoculated to an OD600 of about 0.02 and incubated shaking at 225 rpm, 30° C., until an OD600 of 0.5-0.7 was reached. Cells were then centrifuged at 4° C., washed twice in ice-cold water and once in ice-cold 10% glycerol or 3 mM potassium phosphate (KPi), pH 7.0, before being resuspended in 1/100 of the culture's original volume of 10% glycerol (or 3 mM KPi). Cells were then stored at −80° C. or transformed by electroporation immediately. For transformation, 5 μL (200 ng-2 μg) of plasmid DNA was added to 50 μL of the electrocompetent cells, transferred to a chilled 0.1-cm electroporation cuvette, and electroporated at 1.6 kV, 25 uF, 200 ohms. 450 μL SOC outgrowth medium (NEB) was added to the cells immediately after electroporation and the resuspended cells were incubated shaking at 225 rpm, 30° C., for one hour. The entire transformation was plated on LB agar plates containing appropriate antibiotics (30 μg/mL tetracycline for pCM433-based plasmids, 50 μg/mL kanamycin for pK18mobsacB-based plasmids) and incubated at 30° C. overnight. Transformants were restreaked for single colonies on LB agar and incubated at 30° C. overnight to reduce the possibility of untransformed cells being transferred. For sucrose counter-selection, restreaked transformants were streaked for single colonies on YT+20 or 25% sucrose plates (10 g/L yeast extract, 20 g/L tryptone, 250 g/L sucrose, 18 g/L agar) and incubated at 30° C. overnight. P. putida KT2440 containing the sacB gene can grow, although very slowly, on YT+20% or 25% sucrose media. Therefore, colonies presumed to have recombined the sacB gene out of the genome—those colonies that were larger than most—were restreaked on YT+25% sucrose plates and incubated at 30° C. overnight to reduce the possibility that cells that had not recombined would be carried along with sucrose resistant isolates. Colonies from the second YT+25% sucrose plates were subjected to colony PCR to check for gene replacement at both junctions. These isolates were also plated on LB plates containing appropriate antibiotics to ensure that they had lost antibiotic resistance and, thus, represented pure gene replacements.

Referring to FIGS. 12 a-c, these figures provide experimental data obtained from “proof of concept” tests to show the viability of utilizing engineered microorganisms to metabolize lignin-derived raw materials to produce muconic acid. In this example, alkaline pretreatment with NaOH and anthraquinone (AQ) was applied to corn stover at 70 mg NaOH/g dry biomass with an AQ concentration of 0.2 wt % of dry stover. The resulting alkaline pretreated liquor (APL) stream contains a substantial amount of lignin-derived aromatics such as p-coumarate and ferulate as well as acetate, biomass extractives, and very minor concentrations of sugars (<0.5 g/L of any monomeric sugar). The pH of APL was reduced to about 7 with the addition of H₂SO₄. The liquor was then filtered through a 0.2 μm filter for sterilization and to remove residual solids.

Flasks containing 25 mL of M9 minimal medium supplemented with 0.9×APL were inoculated with P. putida KT2440 or KT2440-CJ103 and cultured for three days. Following biological conversion, cells were removed by centrifugation and activated carbon (12.5 wt/vol %) was added to the remaining culture media to remove non-target aromatics and facilitate analysis by HPLC. Analysis by HPLC detected significant levels of muconic acid in cultures grown with P. putida KT2440-CJ103, while no significant quantities were detected in the blank APL control sample or with the native P. putida KT2440 (see FIG. 12 a). Likewise, analysis of derivatized acids in unpurified culture samples by GCxGC-TOFMS (time-of-flight mass spectrometry) confirmed the identity of muconic acid and displayed comparable trends in concentration, as shown in of FIG. 12 b.

To track the conversion of primary aromatic and nonaromatic components in APL during shake flask cultivation, GCxGC-TOFMS was also employed. Analysis of APL determined that p-coumarate and ferulate were initially present at significant levels (0.92 g/L and 0.34 g/L, respectively), in addition to the short chain acids glycolate and acetate (0.46 g/L and 0.10 g/L, respectively), as shown in FIG. 12 c. Other aromatic acids, including benzoate, caffeate, vanillate, and 4-hydroxybenzoate, were not detected in significant levels (>0.01 g/L). During shake flask cultivations, P. putida KT2440-CJ103 rapidly consumed glycolate and acetate, which can be used as sources of carbon and energy for growth. The primary aromatic components, p-coumarate and ferulate, were converted to 0.70 g/L of muconate after 24 hours (see FIG. 12 c). Based on the consumption of these two major aromatic acids, the molar yield to muconate was 67%.

Example 5 Strains, Media, and Growth Conditions

P. putida KT2440 (ATCC 47054) and its derivatives were grown shaking at 225 rpm, 30° C., in LB Broth or LB plates. During gene replacement, sucrose selection was performed on YT+25% sucrose plates (10 g/L yeast extract, 20 g/L tryptone, 250 g/L sucrose, 18 g/L agar). Shake flask and bioreactor experiments were performed using modified M9 minimal media containing 13.56 g/L disodium phosphate, 6 g/L monopotassium phosphate, 1 g/L NaCl, 2 g/L NH4Cl, 2 mM Mg504, 100 mM CaCl2, and 18 mM Fe504.

Example 6 Shake Flask Experiments with Model Monomers and APL

Fed batch and shake flask experiments were performed using 125 mL baffled flasks containing 25 mL modified M9 media supplemented with 10 mM sodium benzoate, coniferyl alcohol, ferulate, vanillin, caffeate, p-coumarate, 4-hydroxybenzoate or 5 mM phenol and 20 mMsodium acetate or 10 mMglucose. For shake flask experiments in which cells were grown on alkaline pretreated liquor (APL), modified M9 medium was supplemented with APL at a concentration of 0.9×. Cultures were inoculated with cells washed in modified M9 medium to OD₆₀₀ 0.05, then incubated shaking at 30° C., 225 rpm. Every 12 hours cultures were sampled for HPLC, OD₆₀₀, and pH measurement. For cultures at pH>7.4 or <6.6, the pH was adjusted to 7.0 by adding 1 N HCl or 1 N NaOH. 20 mM sodium acetate or 10 mM glucose was added before returning the cultures to the incubator.

Example 7 Fed-Batch Cultivation

A seed batch culture of P. putida KT2440-CJ103 was started in a shake flask and grown overnight in LB, 30° C., 225 rpm. The next morning, cells were centrifuged 3800× g, 10 minutes and washed once with modified M9 medium containing 10 mM glucose. Cultures were transferred to 700 mLs of the same medium in a 2 L Applikon (Applikon Biotechnology, Inc.) EZ Control 2 L bioreactor, starting at an initial OD₆₀₀ of 0.2. Base pH was controlled by 2 N NaOH to pH 7. The temperature was maintained at 30° C. Mixed air was used to deliver oxygen at a flow rate of 2 L/min. DO saturation was manually adjusted to ˜50% by varying stirrer speed, from 250 to 650 rpm, and then maintained at 650 rpm for the duration of the experiment. At 5 hours, 2 mM p-coumarate was added. When glucose was consumed at ˜11.5 h, a large spike in DO was observed, indicating that glucose was depleted and confirmed by YSI analysis. A separate pump was computer programmed to deliver for 30 seconds (˜2.4 mL) a p-coumarate:glucose:ammonium sulfate (68.4:36.5:9 g/L) feed when DOT (dissolved oxygen tension) levels reached ≧75%. The feed caused a temporary drop in DOT to ˜50%, until glucose concentrations fell again. As expected, DOT oscillations proceeded at similar frequencies, until the p-coumarate:glucose:ammonium sulfate feed was terminated at 75.5 hours and the bioreactor was shut down at 78.5 hours.

Muconic Acid Separation, Purification, and Upgrading

Referring again to FIGS. 1 a-c, microbial catalysis operations 140 are only one part of a fully-integrated biorefinery 100. Once a production-scale culture has successfully produced muconic acid, several processing steps remain to obtain a purified muconic acid stream and/or final product(s) that may be manufactured from muconic acid; e.g. adipic acid, nylon-6,6. As discussed previously, one or more separation operations 150 may be required to generate a purified and usable muconic acid stream.

Separation/purification operations 140 are needed to generate a usable muconic acid stream for a number of reasons. For example, a wide variety of impurities may be introduced during the biological production of muconic acid, similar to the challenges faced with other target bio-derived molecules (e.g., ethanol, succinic acid, lactic acid). These impurities may include fermentation salts, nutrients and media to support growth, unconverted substrate, extracellular proteins and lysed cell contents, as well as the buildup of non-target metabolites. Accumulation of these constituents in culture broth may vary greatly depending on the microorganism, substrate used for conversion, biological growth conditions and bioreactor design, and broth pretreatment. Likewise, utilization of monomer streams derived from complex lignocellulosic biomass may vary greatly depending on the biomass fraction of interest (e.g., cellulose, hemicellulose, lignin), choice of feedstock (e.g., herbaceous, hardwoods, softwoods), and depolymerization technology.

Therefore, a culture broth containing a target muconic acid titer will need to be processed before any final products may be manufactured. So, referring to FIG. 13, a muconic-acid containing culture broth 210 may be fed from a bioreactor 200 to a separation/purification operation 150 to produce a purified muconic acid stream of sufficient quality that it may then subsequently be fed to an upgrading 170 section of the biorefinery. Upgrading may include, for example, catalytic hydrogenation of the purified muconic acid to produce adipic acid. The adipic acid may then be further reacted to produce nylon-6,6. In some embodiments of the present invention, the process for producing muconic acid from biomass may further comprise at least one initial separation step that produces the at least one biomass feed stream, wherein the separation step comprises receiving a biomass raw material comprising at least two substituents comprising at least two of lignin, cellulose, hemicellulose, or mixtures thereof, and separating at least two of the substituents to produce the at least one biomass feed stream. In some embodiments of the present invention, the separation separates lignin from polysaccharides to produce a first biomass feed stream comprising lignin, and a second biomass feed stream comprising polysaccharides. The polysaccharide stream may comprise at least one of cellulose, hemicellulose, and mixtures thereof.

Some exemplary technologies that may be utilized in the separation/purification operation 150 include at least one of affinity chromatography, ion exchange chromatography, solvent extraction, liquid-liquid extraction, distillation, filtration, centrifugation, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, differential solubilization, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, and/or reversed-phase HPLC.

Cell removal from the broth may be achieved by a variety of solid removal unit operations. Some examples include filtration, centrifugation, and combinations thereof. Once the microorganism cell matter has been removed, further impurity removal operations may be utilized. For example, the biological ring opening of muconate allows for facile purification from culture media containing non-target aromatic metabolites (e.g., unreacted protocatechuic acid and 4-hydroxybenzoic acid) using activated carbon due to the high adsorption affinity of oxygenated aromatics in comparison to aliphatic acids. FIG. 14 illustrates a comparison of an initial broth (from FIG. 5) and the same broth after treatment with activated carbon. In this example, activated carbon was added to the culture media at about 12.5% (wt/vol) and the resultant mixture was stirred for about 1 hour. This treatment step resulted in nearly complete removal (below detectable limit by HPLC) of protocatechuic acid and 4-hydroxybenzoic acid, while the majority of the muconic acid (89% of initial culture media concentration, mass/vol) remained in solution.

The activated-carbon-treated muconic acid was then crystallized by reducing the pH and temperature, which is enabled due to the strong pH and temperature dependence of dicarboxylic acids. At a pH of about 2 and temperature of about 5° C., muconic acid readily precipitated from solution and the muconic acid crystals were recovered by vacuum filtration. This method recovered 74% of the muconic acid initially present in the activated-carbon-treated muconic acid stream, with a high degree of purity (>97%), as shown in the bottom plot of FIG. 14.

The activated carbon treatment of cell-free, muconic acid containing broth was tested a second time, this time on the broth resulting from culture shown in FIG. 6. The separation process initially consisted of activated carbon purification to remove soluble organic broth impurities and pH/temperature shift crystallization to precipitate muconic acid from purified broth solution and recover solid crystals for drying, similar to what was described above. However, in this example, an additional step, ethanol dissolution and microfiltration was utilized to remove bulk inorganic salts. Purity results for separation tests completed on muconic acid generated by biocatalysis compared to commercial grade muconic acid are summarized in the table below.

TABLE 1 Purity of commercial muconic acid (chemical origin) and biologically derived muconic acid after sequential treatment. Sample AC pH 2 EtOH Origin Treated Crystallized Dissolved Purity (%) Commercial N N N 97.83 ± 0.07% Chemical Biological Y Y N 97.86 ± 0.05% Conversion Biological Y Y Y 99.76 ± 0.04% Conversion ^(a)Standard deviation values reported for triplicate sample measurements.

An activated carbon loading of about 2 wt/vol % was needed to remove residual benzoate from this culture broth to below detectable limits (as determined by high performance liquid chromatography diode array detection. Color compounds in the broth were also removed to a significant extent, turning the broth from a coffee-colored appearance to semi-clear; however, non-selective adsorption of resulted in a 16% reduction in muconic acid broth concentration (6.86 g/L).

Following the activated carbon treatment, muconic acid was precipitated from the broth by pH/temperature shift crystallization. By adjusting the broth pH to 2 with sulfuric acid and reducing the temperature to 5° C., muconic acid readily precipitated. Precipitated muconic acid crystals were then vacuum filtered (0.2-μm PES) and dried in a vacuum oven for about 48 hours. Purity analysis by differential scanning calorimetry (DSC) melting point analysis determined the muconic acid crystals were about 97.83±0.05% pure at this stage. Combustion analysis of muconic acid crystals at 700° C. measured a sample ash content of 1.44% (wt/wt), and elemental analysis by ICP-MS and nitrogen chemiluminescence identified major impurities as sodium (4750 ppm), sulfur (3540 ppm), phosphorus (2860 ppm), potassium (1030 ppm), and nitrogen (336 ppm), as shown in FIG. 15. As shown in FIG. 15, the elemental impurities identified in biologically derived muconic acid differed significantly compared to the impurities observed in chemically derived muconic acid obtained from Sigma Aldrich, with the latter being much lower in sodium, sulfur, phosphorous, potassium, and nitrogen, but higher in iron and chloride. The major inorganic impurities identified in the bio-derived muconic acid are known poisons to platinum group metals, requiring removal strategies prior to catalysis. Likewise, polymer-grade adipic acid requires trace levels of iron (<0.2 ppm) and nitrogen (<20 ppm N), necessitating further treatment.

In order to reduce the level of inorganic impurities in the bio-derived muconic acid and generate a feed stream with sufficient purity and quality to enable successful downstream upgrading, the muconic acid crystals produced in the previous steps were dissolved in ethanol and filtered through a 0.2-μm PES membrane. Upon ethanol dissolution, the muconic acid-ethanol solution was initially cloudy due to insoluble salts, whereas after filtration the solution was very clear (see FIGS. 16 a-f). The initial muconic acid culture broth appeared dark coffee colored (FIG. 16 a). Activated carbon treatment of the broth significantly removed color compounds, while adjustment to a pH of about 2 initiated crystal formation (FIG. 16 b). Filtration and drying of the purified broth produced a white crystal solid, with a purity of 97.86±0.05% by DSC melting point analysis (FIG. 16 c). Muconic acid crystals dissolved in ethanol resulted in a cloudy solution (FIG. 16 d), that upon 0.2-μm microfiltration (FIG. 16 e) resulted in a clear solution (FIG. 16 f) with a final muconic acid purity of 99.76±0.04% upon drying. Analysis of filtered and dried muconic acid after ethanol dissolution revealed an overall DSC-purity of 99.76±0.04% as shown in Table 1 above, with a significant reduction in elemental impurities.

Analysis by ICP-MS determined that sodium was reduced by 96%, sulfur by 99%, phosphorous by 60%, potassium by 82%, and nitrogen by 62% (see FIG. 14), consistent with reductions in low-concentration elements (<100 ppm aluminum, chlorine, magnesium) (results not shown). Biocatalysis-derived muconic acid iron levels were much lower (<1 ppm) compared to muconic acid of chemical origin (141 ppm). Nitrogen levels in bio-muconic acid were still above the polymer precursor specification of 20 ppm, due to residual fermentation proteins that were not removed during activated carbon treatment and microfiltration.

Referring again to FIG. 13, the details provided above demonstrate that biomass-derived muconic acid can be successfully produced by genetically engineered microorganism modified to funnel both lignin depolymerization products and polysaccharide depolymerization products to muconic acid with high yield and selectivity in a bioreactor 200. The muconic acid containing broth 210 can then be successfully treated in a separation/purification operation 150 to produce a purified muconic acid stream 220. This purified muconic acid stream 220 can then be used in an upgrading operation 170 to produce value-added final products, including fuels and commodity chemicals.

As described above, a high volume intermediate that may made from muconic acid is adipic acid, which may then be further converted to nylon-6,6. To evaluate the feasibility of converting bioderived muconic acid to adipic acid, catalyst screening experiments were conducted to identify highly active materials for muconic acid hydrogenation at low temperature and pressure. Commercial noble metal catalysts supported on carbon were initially tested at 5 wt % loading, including palladium, platinum, and ruthenium. Characterization of the virgin catalyst materials (see FIG. 17 a and FIG. 18) revealed the metals were dispersed as small crystallites, with comparable support surface areas (705-1075 m²/g), pore volumes (0.51-0.71 mL/g), and a wider range of exposed active metal areas (22-51% dispersion). Screening experiments found that Pd/C was by the most active catalyst, with consistent activity trends when using M9 culture media (aqueous solution containing salts to support biological growth) or ethanol, as a representative protic polar organic solvent. During the course of the reaction, 2-hexenedioc acid was observed as the primary intermediate, likely due to the low temperature conditions that minimized competing nonselective reaction pathways. For reactions that went to completion with Pd, selectivity to adipic acid was >97% (mol/mol) (See FIG. 17 b).

Additional hydrogenation conditions were examined with Pd/C to (i) determine its activity under surface reaction controlling conditions, (ii) evaluate the apparent activation energy for muconic acid reduction, and (iii) demonstrate its utility with muconic acid recovered from fed-batch biological conversion. Experiments conducted at two different Pd loadings (1 wt % and 2 wt % Pd/C) exhibited comparable turn over frequencies (TOF; 23±6/s and 30±6/s, respectively, at 24 bar of hydrogen and 24° C. in ethanol, 15 mg catalyst, stirring at 1600 rpm), supportive of surface reaction controlling conditions. Experiments to measure the hydrogenation rate of muconic acid at varying temperatures estimated an apparent activation energy of ˜70 kJ/mol (see FIG. 17 c), significantly above values indicative of mass transfer limitation (<20 kJ/mol). Hydrogenation with Pd/C was then demonstrated with muconic acid obtained from fed-batch biological conversion of p-coumarate after activated carbon purification and crystallization. Hydrogenation at room temperature progressed rapidly in a series reaction (see FIG. 17 d, muconic acid TOF 25±3 sec⁻¹), resulting in high purity adipic acid as the final product (>97% mass/mass). After the reaction, analysis of the ethanol solvent indicated that leaching of Pd occurred to a minor extent (7 mg/L, 0.8% of the loaded metal), which may occur due to the acidic liquid phase conditions employed.

Additional studies were completed to evaluate the hydrogenation of bioderived muconic acid to adipic acid. Batch reactor catalyst screening experiments were conducted with platinum group metals to evaluate their activity and stability against leaching during muconic acid hydrogenation. Catalysts were synthesized using powdered Darco activated carbon (AC) and Davisil silica supports sieved to >270 mesh (<53 μm) to minimize the impact of mass transfer during batch conditions. Metals precursors were loaded onto their respective supports, and catalysts were reduced in hydrogen prior to characterization to determine their metal loading and dispersion, support surface area, pore volume and pore diameter, and x-ray diffraction (XRD) spectra, as shown in Table 2 and FIGS. 19 a-j.

TABLE 2 Properties of virgin activated carbon (AC) and silica powdered catalysts used in batch screening reactions for muconic acid hydrogenation. Catalyst S_(BET) Pore vol. ^(a) Pore dia. ^(a) Dispersion ^(b) (nominal) (m² g⁻¹) (cm³ g⁻¹) (Å) (%) 1% Pd/AC 768 0.514 9.71 13 1% Rh/AC 971 0.708 9.83 69 5% Ru/AC 590 0.588 9.69 10 5% Pt/AC 882 0.657 9.71 60 1% Pd/SiO₂ 466 0.774 9.74 28 1% Rh/SiO₂ 480 0.804 9.81 62 5% Ru/SiO₂ 428 0.686 9.75 17 5% Pt/SiO₂ 454 0.811 9.79 47

High surface areas were observed for both AC (590-971 m² g⁻¹) and silica (428-480 m²/g) supported catalysts, with higher metal loading materials generally showing lower surface areas. Support pore volumes (AC 0.514-0.708 cm³/g, silica 0.686-0.811 cm³/g) and pore diameters (AC 9.69-9.83 Å, silica 9.74-9.81 Å) were also comparable. Elemental analysis determined metal loadings were near their nominal values and XRD analysis confirmed the absence of sharp prominent peaks due to large metal crystallites. Chemisorption analysis measured dispersions were within the range of 10-62%, likely due to differing metal precursor and support material interactions during synthesis. Due to varying active metal crystallite surface areas, observed catalyst activities for muconic acid hydrogenation were normalized to dispersion values to allow for turn-over-frequency (TOF) comparisons between metals (e.g., moles of compound reacted per second, divided by the moles of surface metal atoms measured by dispersion).

As shown in FIG. 20 a, pronounced differences were observed in the muconic acid hydrogenation activity between the platinum group metals tested during batch reactor screening experiments (see FIGS. 21 a-h for conversion profiles). Pd and Rh displayed highest TOF for muconic acid hydrogenation on both AC and silica supports, with Pd ranging from 14-23 sec⁻¹ and Rh ranging from 7-8 sec⁻¹. In comparison, Pt displayed a TOF ranging from 1-3 sec⁻, while Ru ranged from 0.4-0.5 sec⁻¹. For batch reactions tested at room temperature, 2-hexenedioic acid was the primary intermediate observed, with trace levels of 3-hexenedioc acid identified on occasion (Scheme 1). For all catalysts screened in the batch system, mass balance closure was typically within +/−10%, with adipic acid as the only product observed upon completion of the reaction.

Scheme 1: Reaction network for the hydrogenation of muconic acid via the intermediate hexenedioic acid (HDA) to produce adipic acid. For room temperature batch reactions, 2-HDA was the primary intermediate, while for higher temperature (≧50° C.) 3-HDA was observed as the primary intermediate.

In addition to differing hydrogenation activity, catalyst metal leaching also varied significantly based on both the metal and choice of support, as shown in FIG. 20 b. Despite exposure to reaction conditions for only 35 min, 1% Pd/SiO₂ displayed the greatest amount of leaching on a percent of initial metal loading basis (8.89%), while 1% Pd/AC leached to a lesser extent (0.93%). Leaching was also observed for 5% Ru/AC (2.93%) and 5% Ru/SiO₂ (3.36%) with muconic acid. In contrast, metal leaching for 1% Rh/AC and 1% Rh/SiO₂ was at trace levels, at 0.51% and 0.16%, respectively, while leaching was negligible for both 5% Pt/SiO₂ (0.04%) and 5% Pt/AC (0.03%). Based on the high activity for muconic acid hydrogenation and stability against leaching, Rh/AC was chosen for further time-on-stream testing in a continuous trickle-bed reactor.

Based on the activity and stability of Rh during batch reactions, continuous trickle bed reactor studies were conducted to determine its 100-h time-on-stream stability, as shown in FIG. 22. Initially, a 1% Rh/AC catalyst was prepared on Norit AC granules crushed and sieved between 100-80 mesh (150-180 μm) to minimize the catalyst bed pressure drop. The catalyst was characterized to determine its virgin and post-reaction properties, as shown in Table 3. Characterization of the virgin 1% Rh/AC catalyst showed high surface area (1029 m² g⁻¹), with comparable pore volume (0.455 cm³ g⁻¹) and average pore diameter (9.69 Å) to the powder Rh/AC catalyst. XRD analysis confirmed the absence of sharp, prominent peaks due to large metal crystallites. Distinct differences were observed in XRD spectra of the powder and granule AC supports, likely due to different vendor carbon sources and/or activation techniques. The metal crystallite dispersion of the granule 1% Rh/AC catalyst was also lower (11.2%) compared to the powder catalyst.

TABLE 3 Properties of virgin and post-reaction 1% Rh/AC granule catalyst used in the 100-h time-on-stream stability test for muconic acid hydrogenation. Catalyst ICP S_(BET) Pore vol. ^(a) Pore dia. ^(a) Dispersion ^(b) (nominal) (%) (m² g⁻¹) (cm³ g⁻¹) (Å) (%) Virgin 0.8 1029 0.46 9.69 14% 1% Rh/AC Post Reaction 0.9 1130 0.52 11.43 21% 1% Rh/AC ^(a) Pore volume and pore diameter (average) determined by BJH desorption. ^(b) Dispersion calculated based on chemisorption and ICP measured metal loading.

The 100-hour time-on-stream stability test of 1% Rh/AC was then evaluated in a sequential fashion, with partial conversion of muconic acid for the first two days to confirm steady state operation, demonstration of complete conversion to adipic acid for days three and four, and lastly a return to partial conversion conditions on day five to observe any changes compared to the initial reactor performance. Sampling of the reactor was not performed during the first 12 hour overnight, since preliminary experiments showed comparable time was required to reach steady conversion once the liquid feed was introduced (see FIG. 23).

During the first 48-hours of time-on-stream (50° C., 0.5 mL min⁻¹ liquid flow rate), muconic acid was partially converted (57.7±1.9% average molar conversion) to hexenedioic acid (HDA) and adipic acid as the only observed products. Product identities were confirmed by gas chromatography mass spectroscopy. The moderately higher reaction temperature (50° C.) resulted in isomerization to 3-HDA as the predominant species (30.9±1.2% molar yield), in comparison to 2-HDA for room temperature batch screening reactions. Moderate amounts of 2-HDA (19.7±2.9% molar yield) and adipic acid (9.7±1.2% molar yield) were also produced, with an average molar closure of 102.7±4.9%, supporting steady state conversion during the first 48 hours. Variability in molar closure was assumed to be primarily due to solvent evaporation and error introduced during the sampling of knockout pot, with concentrations of individual species throughout the 100-h run reported in Table 4 below.

TABLE 4 Time-on-stream results for the trickle bed hydrogenation of muconic acid.^(a) Compounds were monitored by HPLC-RID. Temp Liq. flow Muconic 2-HDA 3-HDA Adipic Time (h) (° C.) (mL/min) (g/L) (g/L) (g/L) (g/L) 18 50 0.5 2.98 1.30 2.50 0.79 20 50 0.5 3.12 1.26 2.48 0.77 22 50 0.5 3.20 1.16 2.35 0.72 24 50 0.5 3.44 1.78 2.60 0.83 37 50 0.5 3.21 1.68 2.40 0.78 39 50 0.5 3.36 1.64 2.34 0.74 41 50 0.5 3.38 1.69 2.40 0.77 43 50 0.5 3.31 1.67 2.34 0.79 45 50 0.5 3.31 1.65 2.34 0.75 67 78 0.2 0.00 0.00 0.00 8.78 69 78 0.2 0.00 0.00 0.00 7.97 71 78 0.2 0.00 0.00 0.00 8.14 73 78 0.2 0.00 0.00 0.00 8.02 88 78 0.2 0.00 0.00 0.00 7.92 92 78 0.2 0.00 0.00 0.00 7.97 96 78 0.2 0.00 0.00 0.00 8.14 114 50 0.5 3.63 1.45 1.99 0.78 116 50 0.5 3.70 1.80 1.91 1.00 118 50 0.5 3.38 1.71 2.07 1.14 120 50 0.5 3.10 1.63 2.03 0.89 ^(a)Reaction conditions were as follows: Muconic acid 1 wt % in ethanol, liquid flow rate and temperature as indicated, H₂ flow 200 sccm, system pressure 24 bar, 1100 mg 1% Rh/AC granules.

Multiple factors can influence the observed reaction rates in trickle bed reactors, including the gas-liquid flow rate ratio, liquid film thickness due to shear, interparticle and intraparticle wetting, and catalyst particle size, shape, and packing geometry. Based on the liquid feed rate flow rate and conversion observed during the first 48 hours, the muconic acid hydrogenation TOF was calculated to be 0.022 sec⁻¹ at 50° C., which was ˜ 1/1000^(th) of the rate observed for powder Rh/AC in batch reactor screening experiments at 24° C. (TOF 7 sec⁻¹), indicating external and intraparticle diffusion likely influenced the observed rate due to larger particle sizes required for trickle-bed reactor experiments. Varying the catalyst bed temperature from 50-72° C. resulted in an apparent activation energy of 60.7 kJ mol⁻¹ for the 1% Rh/AC granule catalyst, well above typical barriers observed under solely mass transfer limiting conditions (<20 kJ mol⁻¹) and comparable to batch reactor results for powder 1% Pd/AC (70 kJ/mol). However, the focus was on examining alterations to the catalyst material properties after time-on-stream rather than a detailed kinetic analysis.

To demonstrate complete conversion of muconic acid to adipic acid, the temperature was increased and liquid flow rate reduced (78° C., 0.2 mL min⁻¹) for day three and four of operation. No peaks from HDA were observed by HPLC-DAD, which was highly sensitive to the presence of olefin bonds, supporting near complete conversion of muconic acid to adipic acid. Lastly, reaction conditions were returned to partial conversion conditions for day five to compare the catalyst conversion and selectivity to the first 48 hour of time-on-stream. Mass balance and product distribution perturbations were observed when altering the liquid flow rate, with a trend toward increasing conversion as time continued. For day five, the average muconic acid molar conversion was 55.2±3.6%, comparable to values observed during the first 48 h of time-on-stream (57.7±1.9%). Product distribution molar yields were also comparable, with average molar closure of 103.5±4.6%.

The following provides further disclosure regarding the solid catalysts describe above for the hydrogenation reaction to convert muconic acid to adipic acid. As used herein, “solid” refers to a solid material that is used as a catalyst and/or as a physical support for one or more catalytic elements. Thus, a solid may provide catalytic activity itself, may provide a structure upon which to build and physically support catalytic elements, or both. Examples of solids used in some embodiments of the present invention include, but are not limited to, carbonaceous materials, oxides, polymers, carbonates, sulfates, and clays. A non-limiting example of a carbonaceous solid is activated carbon. Examples of oxide solids include, but are not limited to, alumina, silica, titanium dioxide, and aluminosilicates. In some embodiments, a carbonaceous material or a silica-containing material may be used as a solid support and/or a solid catalyst for the catalytic conversion of muconic acid to adipic acid.

As used herein, “active site” or “active material” refers to a physical and/or chemical feature that catalyzes a reaction. A catalyst is a substance, structure, element, composition, compound, molecule, or combination thereof that accelerates a chemical reaction without itself being consumed. Examples of active sites include, but are not limited to, one or more elements in their pure form, or in mixtures to form covalently bond molecules, salts, ions, and mixtures thereof. Thus, catalytic active sites may be placed on a solid material. Such active sites may be incorporated into the solid structure itself, for example, by reaction to form covalent bonds that chemically attach at least some of the active sites to the solid. In some embodiments, at least one metal may be combined with a solid to provide a catalyst for the conversion of muconic acid to adipic acid.

In some embodiments, a metallic catalyst comprising a solid may include at least one active site either incorporated into the solid or deposited on the solid, wherein the at least one active site is at least one noble metal, or mixtures thereof. In some cases, a metallic catalyst including a solid may have at least two active sites either incorporated into the solid or deposited on the solid, wherein the at least two active sites are at least two noble metals, or mixtures thereof. As used herein, a “noble metal” refers to at least one of ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, and/or gold. In some further embodiments, a metallic catalyst that includes a solid may have at least one active site either incorporated into the solid or deposited on the solid, wherein the at least one active site is at least one of a noble metal, mercury, rhenium, and/or copper. In some examples, a metallic catalyst including a solid may have at least two active sites either incorporated into the solid or deposited on the solid, wherein the at least two active sites are at least two of a noble metal, mercury, rhenium, and/or copper.

A metallic catalyst that includes a solid may be constructed from solid carbon and at least one of palladium, platinum, and/or ruthenium. At least one metal of a bimetallic catalyst may be at least one of palladium, platinum, and/or ruthenium, may be present in metallic form and/or as a salt. Palladium may be in the 0, +1, +2, +3, +4 oxidation state, or mixtures thereof. Platinum may be in the 0, +1, +2, +3, +4 oxidation state, or mixtures thereof. Ruthenium may be in the −2, 0, +1, +2, +3, +4, +5, +6, +7, +8 oxidation state, or mixtures thereof. Furthermore, a solid used as a support for a metallic catalyst may be at least one of carbon nanotubes, graphene, and/or activated carbon. A bimetallic catalyst may be constructed from intermetallic/core shell nanoparticles. Thus, a bimetallic catalyst comprising two metals and a solid may be utilized to catalyze the hydrogenation reaction of cis,cis-muconic acid with diatomic hydrogen to form at least one of adipic acid, 1,6-hexanediol, or mixtures thereof.

Example 8 Catalyst Synthesis

Commercial monometallic noble metal catalysts were screened for their hydrogenation activity with muconic acid. Catalysts at 5 wt % loading on activated carbon were obtained from Sigma Aldrich (Pt, Pd, and Ru) and 1 wt % Pd/C was obtained from Alfa Aesar. Virgin catalyst materials were initially characterized to determine their average crystallite size and long-range order by X-ray diffraction, support surface area and pore volume by nitrogen physisorption, and active metal surface area by hydrogen chemisorption, with details described elsewhere. Due to the high sensitivity of Pd dispersion with temperature, Pd samples were reduced under flowing hydrogen (50 mL/min, 10% H₂ in Ar) at moderate temperature (125° C., 3° C./min) and held for 1 hour. Following reduction, Pd samples were purged for 1 hour under Ar and cooled to 45° C. prior to H₂/O₂ titration. For calculations of Pd dispersion, the amount of hydrogen uptake that followed the second oxygen titration was used. A stoichiometry of 0.667 Pd sites per H₂ molecule was assumed to remove oxidized Pd—O species in the form of water and form the reduced Pd—H species.

Example 9 Catalyst Synthesis

Platinum group metal catalysts (Pt, Rh, Ru, Pd) were synthesized on powder carbon and silica supports to evaluate their activity and stability for muconic acid hydrogenation. For batch reaction studies, Darco activated carbon (Sigma Aldrich) and Davisil Grade 633 high surface area silica (Sigma Aldrich) were used. Supports were initially sieved >270 mesh (<53 μm) to minimize the impact of mass transfer on observed kinetics. The silica support was calcined at 500° C. in air prior to loading metals, while the activated carbon support was used as received. Catalysts were prepared with the following metal salt precursors: palladium acetate (Sigma Aldrich), rhodium nitrate hydrate (Sigma Aldrich), ruthenium chloride hydrate (Sigma Aldrich), chloroplatinic acid (CPA) (Sigma Aldrich), and ammonium tetraammineplatinum nitrate (PTA) (Sigma Aldrich). Pd, Ru, and Rh catalysts were prepared by incipient wetness, while Pt catalysts were prepared by strong electrostatic adsorption (SEA) to improve dispersion due to the low activity. For SEA catalyst synthesis, 1.9 g of support was added to 50 mL of DI water, and the pH was adjusted to facilitate protonation/deprotonation of the support (pH 12 with NaOH for silica, pH 2.9 with HCl for AC). In another bottle, the appropriate catalyst precursor was dissolved in 50 mL of DI water (PTA for silica, CPA for activated carbon). The two bottles were mixed together with stirring for 1 hour, followed by vacuum filtration to recover the catalyst. The catalyst was washed twice with 50 mL of DI water and left to dry overnight in air at room temperature. After loading, catalysts were dried at 110° C. and reduced in hydrogen flowing at 200 sccm for 2 hours at temperature. Due to the sensitivity of Pd dispersion with temperature, Pd catalysts were reduced at 125° C. while Pt, Rh, and Ru catalysts were reduced at 250° C.

For flow reactor studies, extruded activated carbon pellets (Norit Rx 3 Extra, Cabot Norit) were initially crushed and sieved between 80-100 mesh (150-180 μm) to allow for a moderate catalyst bed pressure drop (<5 psig) while still facilitating mass transfer. Rh was loaded onto the support by incipient wetness using ruthenium chloride hydrate (Sigma Aldrich), dried at 110° C., and reduced ex situ prior to use at 250° C. in flowing hydrogen.

Catalysts were characterized after synthesis and reduction to determine their virgin properties, as well as post-reaction for flow reactor experiments. X-ray diffraction (XRD) was used to assess catalyst metal crystallite size and bulk long-range order. Catalyst support surface area, pore volume, and average pore diameter were measured by BET nitrogen physisorption. Scanning electron microscopy, coupled to energy dispersive electron spectroscopy, was used to evaluate of metal crystallite distribution on the support. Chemisorption was used with to evaluate crystallite metal dispersion, defined as the percentage of metal surface sites compared to the total metal loaded.

Example 10 Catalyst Testing

For batch reactor activity studies, reactions were performed in using a Para 5000 Multi-reactor system (Parr Instruments). Commercial cis,cis-muconic acid in the amount of 200 mg (Sigma Aldrich) was dissolved in 19.8 g of 200 proof ethanol. The muconic acid solution and 15 mg of catalyst were then loaded into 75-mL vessels equipped with magnetic stirring. Hydrogenation reactions were performed at 24° C. with hydrogen supplied at a constant 24 bar and stirring at 1600 rpm. Duplication reactions were performed at minimum, with error bars indicating sample standard deviations. Samples were collected via an in situ sample port, syringe filtered, and analyzed by HPLC, as described below. After the reaction, the reactor contents were vacuum filtered (0.2-μm PES filter assembly, Nalgene) to remove catalyst particles, and subsequently the liquid filtrate was analyzed by ICP-OES to examine active metal leaching.

For flow reactor stability studies, reactions were performed using a Parr Tubular reactor system (Parr Instruments) operated in a down-flow trickle-bed configuration. The system was outfitted with a HPLC pump to deliver liquid phase reactants (Series III Scientific Instrument), pair of mass flow controllers to control inert and hydrogen gas delivery (Brooks), tube-in-tube heat exchanger for cooling the reactor effluent, high-pressure 1-L stainless steel knockout pot with bottom sampling valve, and a solenoid-controlled backpressure regulator (Tescom) to maintain system pressure. Reactions were performed in trickle down flow configuration, with gas and liquid reagents fed to through the top of a 32″ long, ¼″ inner diameter stainless steel reaction tube. The tube temperature was monitored and controlled using an internal thermocouple centered in the catalyst bed and three furnace wall thermocouples. The tube was initially packed halfway with inert 1-mm glass beads (Sigma Aldrich) held in place with quartz wool (Quartz Scientific Inc.). The catalyst bed was then loaded at the tube mid-height. Inert quartz sand (Quartz Scientific Inc.) sieved to <60 mesh (>250 μm) was placed at the base and top of the carbon catalyst packing to serve as a support. The remaining reactor tube void was then filled with inert glass beads and sealed with quartz wool.

Continuous hydrogenation reactions were performed with hydrogen supplied at 200 sccm and a system pressure maintained at 24 bar. Temperature was varied from 50-78° C., as indicated. The mobile phase consisted of biologically derived muconic acid purified with activated carbon, precipitated by temperature-pH shift crystallization, dissolved in 200-proof ethanol (8 g/L), and filtered (0.2-μm PES) to remove insoluble salts. Commercial succinic acid (Sigma Aldrich, ≧99.0% reagent purity) was added as an internal standard (0.8 g/L). The liquid flow rate was varied from 0.2-0.5 mL/min, as indicated. Liquid r eactor effluent samples collected from the knockout pot were syringe-filtered, and analyzed by HPLC and GC-MS, as described below. Periodically, the liquid filtrate was analyzed by ICP-MS to detect catalyst metal leaching. After testing, the reactor was cooled to room temperature, depressurized, and 500 mL of ethanol was flushed through the catalyst bed, followed by drying under 200 sccm nitrogen. The catalyst bed packing solids were then sieved between 80-100 mesh (150-180 μm) to recover the catalyst granules for further analysis.

Nylon-6,6 Polymerization with Bio-Adipic Acid

Bio-adipic acid produced from muconic acid was then polymerized with 1,6-hexanediamine to form nylon-6,6 for comparative material testing to petrochemical adipic acid. Bench-scale condensation polymerizations were conducted using the nylon rope reaction shown in FIG. 24. After producing nylon fibers from both bio-adipic acid and commercial adipic acid, polymer materials were dried and characterized to determine their thermal and physicochemical properties.

Thermal analysis of both nylon materials by DSC showed comparable melting and glass transition temperatures, similar to values reported in literature for nylon-6,6, as shown in Table 5 below. Clean thermal traces were observed for nylon produced from biologically derived adipic acid, with a heat of fusion comparable (50.2 J/g) to literature values (51.3 J/g).

Measurement of the intrinsic viscosity by dilute solution viscometry showed similar values for the two nylon materials, and calculations of the viscosity average MW showed that polymerization had taken place to a comparable extent for bio-adipic acid (1,920±20 g/mol) and chemical adipic acid (2,230±40 g/mol). However, the limitation of the nylon rope trick was apparent for achieving industrially relevant nylon MW values (40,000-60,000 g/mol).

TABLE 5 Properties of nylon-6,6 produced using commercial adipic acid of chemical origin and bio-adipic acid generated in this work from the catalytic hydrogenation of muconic acid. Nylon-6,6 Adipic Acid Adipic Acid Literature for Properties Chemical Biological Nylon-6,6 Melting Temp 258 264 262 (° C.) Glass Transition 55 46 50 (° C.)^(a) Heat of fusion 37.8 50.2 51.3 (J/g) Crystallinity 19.9 26.4 27.0 (%) Intrinsic Viscosity 26.5 ± 0.9  24.1 ± 0.6   79−174 (mL/g)^(b) Viscosity Avg. MW 2,230 ± 40   1,920 ± 20   40,000−60,000 (g/mol)^(b) ^(a)T_(g) determined from literature for 27% crystallinity and is known to vary. ^(b)Standard deviation values reported for four solutions tested in triplicate.

FIG. 25 illustrates a process flow diagram of one example of a portion of a biorefinery, downstream of the biocatalytic bioreactor corresponding to the muconic acid separation/purification and upgrading portions. Thus, FIG. 25 summarizes one hypothetical process and these separation/purification and upgrading steps to convert culture broth from a bioreactor to adipic acid.

Referring to FIG. 25, Area 100 of the process model focuses on purification and recovery of muconic acid from the biological culture broth. Muconic acid broth may be purified over parallel activated carbon treatment beds to remove non-target aromatic impurities. Spent activated carbon from purification may be thermally regenerated onsite by kiln combustion due to the high boiling point of adsorbed organics. After purification, low pH and low temperature crystallization may be employed to recover muconic acid based on a solubility of 3.5 g/L at pH 2 and 10° C. Concentrated sulfuric acid may be used for pH adjustment, producing Na₂SO₄ (2 g of Na₂SO₄ per 1 g of muconic acid solid) that co-crystallizes at a solubility limit of 82.8 g/L at 10° C. Rotary filtration and rotary drying may then be employed to recover mixed solid crystals, with the filter broth effluent treated as wastewater. Mixed solid crystals may be added to ethanol in a heated stirred tank at 70° C. to dissolve muconic acid, while insoluble Na₂SO₄ may be separated by rotary filtration and treated as solid waste.

Area 200 shown in FIG. 25 focuses on the purification and catalytic conversion of muconic acid to adipic acid, with subsequent product recovery. Muconic acid in ethanol may be initially pressurized to 350 psig using a positive-displacement pump for feeding to the reactor. On site hydrogen may be supplied at ambient temperature and pressure, utilizing a 3-stage compression train with inter-stage cooling to deliver hydrogen at 350 psig. Muconic acid, ethanol, and hydrogen may then be mixed and introduced to a trickle bed reactor operating at 70° C. with a weight hour space velocity (WHSV), defined as the weight of liquid solution processed per hour divided by the weight of catalyst material, of 5 h⁻¹. Hydrogenation of muconic acid may be achieved utilizing a 2% Rh/C catalyst, to produce substantially pure adipic acid.

Following hydrogenation, adipic acid may be recovered from solution by ethanol evaporation and crystallization. The stream of adipic acid in ethanol exiting the reactor may be mixed with the crystallizer recycle stream and concentrated to 360 g/L at 82° C., below the adipic acid/ethanol solubility limit of 363 g/L at 60° C. The solution may then be cooled to 10° C. to partially crystallize adipic acid based on a solubility limit of ˜67 g/L at 10° C., with the remaining solution recycled to the inlet of the evaporator. Rotary filtration and drying may then be employed to dry crystals, with an assumed net adipic acid recovery of about 98% post-hydro genation.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope. 

What is claimed is:
 1. A genetically modified microorganism comprising at least one exogenous gene addition, wherein the at least one added gene encodes at least one of a decarboxylase, a dehydratase, or a monooxygenase.
 2. The microorganism of claim 1, further comprising at least one endogenous gene deletion, wherein the at least one deleted gene encodes at least one of a dioxygenase, a muconate lactonizing enzyme, or muconolactone isomerase.
 3. The microorganism of claim 1, wherein the microorganism over-expresses at least one demethylase gene.
 4. The microorganism of claim 1, further comprising a deletion of at least one catabolite repression control gene of the microorganism.
 5. The microorganism of claim 1, wherein the at least one exogenous gene encodes a decarboxylase from Enterobacter cloacae.
 6. The microorganism of claim 5, wherein the exogenous gene is at least one of aroY, ecdB, or ecdD.
 7. The microorganism of claim 1, wherein the at least one exogenous gene encodes a dehydratase from Bacillus cereus.
 8. The microorganism of claim 7, wherein the exogenous gene is at least one of aroZ or asbF.
 9. The microorganism of claim 1, wherein the at least one exogenous gene encodes a monooxygenase from Pseudomonas sp. CF600.
 10. The microorganism of claim 9, wherein the exogenous gene is at least one of dmpK, dmpL, dmpM, dmpN, dmpO, dmpP, or pheA.
 11. The microorganism of claim 2, wherein the at least one deleted gene is at least one of pcaH or pcaG.
 12. The microorganism of claim 2, wherein the at least one deleted gene is at least one of catB or catC.
 13. The microorganism of claim 3, wherein the demethylase gene is at least one of vanA, vanB, or ligM.
 14. The microorganism of claim 1, wherein the microorganism is at least one of a bacterium, a fungus, a yeast, a prokaryote, or a prokaryotic microorganism.
 15. The microorganism of claim 14, wherein the microorganism is a prokaryote or prokaryotic microorganism from the genus Pseudomonas.
 16. The microorganism of claim 15, wherein the microorganism is a strain of P. putida, P. fluorescens, or P. stutzeri.
 17. The microorganism of claim 16, wherein the microorganism is a strain of P. putida KT2440.
 18. A process for producing muconic acid, the process comprising contacting a culture broth containing lignin depolymerization compounds with the microorganism of claim
 1. 19. The process of claim 18, wherein the lignin depolymerization compounds comprise at least one of p-coumaric acid, ferulic acid, benzoic acid, phenol, coniferyl alcohol, caffeic acid, vanillin, or 4-hydroxybenzoic acid, and at least a portion of the lignin depolymerization compounds are converted to catechol, and at least a portion of the catechol is converted to muconic acid.
 20. A process for producing adipic acid, the process comprising: separating muconic acid from a culture broth comprising muconic acid, impurities, and a microorganism; purifying the separated muconic acid; and hydrogenating at least a portion of the purified muconic acid to produce the adipic acid.
 21. The process of claim 20, wherein the separating comprises at least one of centrifugation or filtration to produce muconic acid that is substantially free of the microorganism.
 22. The process of claim 21, wherein the purifying comprises contacting the separated muconic acid with an adsorbent, wherein the adsorbent removes at least a first portion of the impurities from the separated muconic acid.
 23. The process of claim 22, wherein the adsorbent comprises activated carbon.
 24. The process of claim 23, wherein the impurities removed comprise at least one of benzoic acid, protocatechuic acid or 4-hydroxybenzoic acid.
 25. The process of claim 20, wherein the purifying comprises crystallizing at least a portion of the muconic acid from the separated muconic acid to form a muconic acid precipitate and a liquid that contains at least a portion of the impurities.
 26. The process of claim 25, wherein the purifying further comprises: dissolving the muconic acid precipitate in a solvent, resulting in a liquid phase comprising muconic acid and a solid phase comprising at least a portion of the impurities; and separating the liquid phase from the solid phase.
 27. The process of claim 26, wherein the separating is by at least one of filtration or centrifugation.
 28. The process of claim 26, wherein the hydrogenation comprises contacting the liquid phase comprising muconic acid and diatomic hydrogen with a metallic catalyst.
 29. The process of claim 28, wherein the metallic catalyst comprises at least one of palladium, platinum, ruthenium, or rhodium.
 30. The process of claim 29, wherein the at least one of palladium, platinum, ruthenium, or rhodium is supported by activated carbon or silica.
 31. The process of claim 30, wherein the metallic catalyst comprises rhodium supported by activated carbon. 